Factor IX polypeptides and methods of use thereof

ABSTRACT

The present invention provides methods of administering Factor IX; methods of administering chimeric and hybrid polypeptides comprising Factor IX; polynucleotides encoding such chimeric and hybrid polypeptides; cells comprising such polynucleotides; and methods of producing such chimeric and hybrid polypeptides using such cells.

REFERENCE TO EARLIER FILED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/809,276, filed Jul. 11, 2011, which is the national phaseapplication of International Application No. PCT/US2011/043569, filedJul. 11, 2011 and published as WO 2012/006624, which claims the benefitof U.S. Provisional Application No. 61/363,064, filed Jul. 9, 2010, U.S.Provisional Application No. 61/424,555, filed Dec. 17, 2010, U.S.Provisional Application No. 61/430,819, filed Jan. 7, 2011, U.S.Provisional Application No. 61/438,572, filed Feb. 1, 2011, U.S.Provisional Application No. 61/442,079, filed Feb. 11, 2011, and U.S.Provisional Application No. 61/470,951, filed Apr. 1, 2011, all of whichare incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:Substitute_Sequence_Listing21592730007.txt, Size: 20,416 bytes; and Dateof Creation: Jul. 10, 2015) submitted in this application isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of therapeutics forhemostatic disorders.

2. Background Art

Hemophilia B (also known as Christmas disease) is one of the most commoninherited bleeding disorders in the world. It results in decreased invivo and in vitro blood clotting activity and requires extensive medicalmonitoring throughout the life of the affected individual. In theabsence of intervention, the afflicted individual will suffer fromspontaneous bleeding in the joints, which produces severe pain anddebilitating immobility; bleeding into muscles results in theaccumulation of blood in those tissues; spontaneous bleeding in thethroat and neck may cause asphyxiation if not immediately treated; renalbleeding; and severe bleeding following surgery, minor accidentalinjuries, or dental extractions also are prevalent.

Normal in vivo blood coagulation at minimum requires the serineproteases Factors II (prothrombin), VII, IX, X and XI (soluble plasmaproteins); cofactors including the transmembrane protein tissue factorand the plasma proteins Factors V and VIII; fibrinogen, thetransglutaminase Factor XIII, phospholipid (including activatedplatelets), and calcium. Additional proteins including kallikrein, highmolecular weight kininogen, and Factor XII are required for some invitro clotting tests, and may play a role in vivo under pathologicconditions.

In hemophilia, blood clotting is disturbed by a lack of certain plasmablood clotting factors. Hemophilia B is caused by a deficiency in FactorIX that may result from either the decreased synthesis of the Factor IXprotein or a defective molecule with reduced activity. The treatment ofhemophilia occurs by replacement of the missing clotting factor byexogenous factor concentrates highly enriched in Factor IX. However,generating such a concentrate from blood is fraught with technicaldifficulties, as is described below.

Purification of Factor IX from plasma (plasma derived Factor IX; pdFIX)almost exclusively yields active Factor IX. However, such purificationof factor IX from plasma is very difficult because Factor IX is onlypresent in low concentration in plasma (5 ug/mL. Andersson, ThrombosisResearch 7: 451 459 (1975). Further, purification from blood requiresthe removal or inactivation of infectious agents such as HIV and HCV. Inaddition, pdFIX has a short half-life and therefore requires frequentdosing. Recombinant factor IX (rFIX) is also available, but suffers fromthe same short half-life and need for frequent dosing (e.g., 2-3 timesper week for prophylaxis) as pdFIX. rFIX also has a lower incrementalrecovery (K value) compared to pdFIX, which necessitates the use ofhigher doses of rFIX than those for pdFIX.

Reduced mortality, prevention of joint damage and improved quality oflife have been important achievements due to the development ofplasma-derived and recombinant Factor IX. Prolonged protection frombleeding would represent another key advancement in the treatment ofhemophilia B patients. However, to date, no products that allow forprolonged protection have been developed. Therefore, there remains aneed for improved methods of treating hemophilia due to Factor IXdeficiency that are more tolerable and more effective than currenttherapies.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of administering Factor IX usingchimeric polypeptides comprising Factor IX and hybrids of such chimericpolypeptides; chimeric polypeptides comprising Factor IX and hybrids ofsuch chimeric polypeptides; polynucleotides encoding such chimeric andhybrid polypeptides; cells comprising such polynucleotides; and methodsof producing such chimeric and hybrid polypeptides using such cells. Insome embodiments, the Factor IX chimeric polypeptide is a Factor IX FcRnbinding partner (BP) chimeric polypeptide such as a Factor IX Fcchimeric polypeptide. In other embodiments, the Factor IX chimericpolypeptide is a Factor IX-XTEN polypeptide.

The present invention provides a method of administering Factor IX to asubject in need thereof, comprising administering to the subject a doseof at least about 10, at least about 20, or at least about 25 IU/kg of aFactor IX FcRn BP chimeric polypeptide, e.g., a Factor IX-Fc chimericpolypeptide or a Factor IX-XTEN chimeric polypeptide, at about a onceweekly or longer dosing interval.

In some embodiments, the plasma level of the chimeric polypeptidereaches an average trough of at least about 1 IU/d after at least about6 days in at least about 70%, at least about 80%, at least about 90%, orabout 100% of a patient population or reaches a trough of at least about1, 2, 3, 4, or 5 IU/dl after at least about 6 days in a subject. In someembodiments, the plasma level of said chimeric polypeptide reaches anaverage trough of about 1-5 or 1-3 IU/dl. Such trough or average troughmay be reached after about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, or about 40 days.

In some embodiments, the chimeric polypeptide has greatly reducedphosphorylation and sulfation in comparison to plasma derived Factor IX.In some embodiments the chimeric polypeptide is less than 25%phosphorylated and less than 25% sulfated, e.g., less than 25% fullyphosphorylated and sulfated. In some embodiments, the chimericpolypeptide is less than about 10% phosphorylated and less than about 9%sulfated. In some embodiments, the chimeric polypeptide has a gammacarboxylation pattern/distribution, a gamma carboxylation content, asialylation pattern/distribution, and/or a sialylation content similarto (i.e., within 10% of) or the same as those of the Factor IX Fcchimeric polypeptide in Examples 5-6.

In some embodiments, the chimeric polypeptide has an incrementalrecovery greater that 0.7 or greater than 0.75 ug/ml (antigen). In someembodiments, the chimeric polypeptide has a mean incremental recovery(K-Value) (activity; observed) of at least about 0.8, at least about0.9, or at least about 1 IU/dL per IU/kg.

In some embodiments, the chimeric polypeptide exhibits one or morepharmacokinetic parameters, in said patient population or in saidsubject, selected from the group consisting of:

(a) a mean clearance (CL) (activity) in said patient population of about3.36±0.93 mL/hour/kg; a mean clearance (CL) (activity) in said patientpopulation of about 3.0-3.72, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, or3.72 mL/hour/kg; a mean clearance (CL) (activity) in said patientpopulation that is about 2.5 fold lower than the clearance of apolypeptide comprising said Factor IX without said FcRn BP; a clearance(CL) (activity) in said subject of about 1.84-4.58 mL/hour/kg

(b) a mean residence time (MRT) (activity) in said patient population ofat least about 68.05±11.16 hours; a mean MRT (activity) in said patientpopulation of about 60-78, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78hours; a mean MRT (activity) in said patent population that is about 3fold longer than the mean MRT of a polypeptide comprising said Factor IXwithout said FcRn BP; a mean residence time (MRT) (activity) in saidsubject of about 53.1-85.8 hours; a mean residence time (MRT) (activity)in said subject of at least about 45, about 50, about 55, about 60,about 65, about 70, about 75, about 80, about 85, or about 90 hours;

(c) a mean t_(1/2beta) (activity) in said patient population of about52.5±9.2 hours; a mean t_(1/2beta) (activity) in said patient populationthat is about 47-60 hours, about 47, about 48, about 49, about 50, about51, about 52, about 53, about 54, about 55, about 56, about 57, about58, about 59, about 60 hours; a mean t_(1/2beta) (activity) in saidpatient population that is about 3 fold longer than the mean t_(1/2beta)of a polypeptide comprising said Factor IX without said FcRn BP; at_(1/2beta) (activity) in said subject of about 40-67.4, about 40, about45, about 50, about 55, about 60, about 65, about 70, or about 75 hours;

(d) a mean incremental recovery (K value) (activity; observed) in saidpatient population of about 0.93±0.18 IU/dL per IU/kg; a meanincremental recovery (K value) (activity; observed) in said patientpopulation of about 0.85-1.0, about 0.85, about 0.86, about 0.87, about0.88, about 0.89, about 0.90, about 0.91, about 0.92, about 0.93, about0.94, about 0.95, about 0.96, about 0.97, about 0.98, about 0.99, about1.0, about 1.05, about 1.10, or about 1.15 IU/dL per IU/kg; a meanincremental recovery (K value) (activity; observed) in said patientpopulation that is about 24% better than the mean incremental recoveryof a polypeptide comprising said Factor IX without said FcRn BP; anincremental recovery (K value) (activity; observed) in said subject ofabout 0.62-1.17 IU/dL per IU/kg;

(e) a mean Vss (activity) in said patient population of about 226±67.76(corrected to 69.8) mL/kg; a mean Vss (activity) in said patientpopulation of about 200-300, about 200, about 210, about 220, about 230,about 240, about 250, about 260, about 270, about 280, about 290, orabout 300 mL/kg; a Vss (activity) in said subject of about 145-365mL/kg;

(f) a mean AUC/dose (activity) in said patient population of about32.44±10.75 IU*h/dL per IU/kg; a mean AUC/dose (activity) in saidpatient population of about 26-40, about 26, about 27, about 28, about29, about 30, about 31, about 32, about 33, about 34, about 35, about36, about 37, about 38, about 39, or about 40 IU*h/dL per IU/kg; anAUC/dose in said subject of about 21.80-54.30 IU*h/dL per IU/kg.

In some embodiments, the dose of chimeric polypeptide contains asignificantly lower (10-100 fold) level (0.01-0.001%) of activated FIX(FIXa), than currently marketed Factor IX products such as MONONINE™(pdFIX; CSL Behring)) or BENEFIX™ (Wyeth; rFIX) (0.1%). Such level maybe 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold lower than currentlymarketed products, or 0.01, 0.05, 0.0033, 0.0025, 0.002, 0.00167,0.00142, 0.00125, 0.00111, or 0.001%.

In some embodiments, the dosing interval is 6-18, 6-10, 9-18, at least6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, or at least 18 days, weekly, two times monthly, or one time monthly.The dosing interval may be a prophylactic dosing interval, a fixedprophylactic dosing interval, or an individualized prophylactic dosinginterval.

The methods of the invention are practiced on a subject in need ofcontrol or prevention of bleeding or bleeding episodes, in need ofintermittent treatment, in need of prophylactic treatment, or in need ofon-demand treatment.

The therapeutic doses that may be used in the methods of the inventionare about 25-180, about 20-180, about 20-50, about 20-100, about 10-180,about 10-50, about 10-30, or about 50-100 IU/kg. The dose may be a fixeddose or an individualized dose.

In some embodiments, the chimeric polypeptide is administeredintravenously or subcutaneously.

The subject in the methods of the invention may be a human subject ormay be a non-human mammal. Non-human mammals include mice, dogs,primates, monkeys, cats, horses, cows, pigs, and other domestic animalsand small animals.

The chimeric polypeptide may be in the form of a hybrid comprising asecond polypeptide in association with said chimeric polypeptide,wherein said second polypeptide comprises or consists essentially of anFcRn BP, e.g., an Fc. The chimeric polypeptide may be at least 90%, atleast 95%, or 100% identical to the Factor IX sequence, the Fc sequence,or both the Factor IX and Fc sequence in Tables 2A (SEQ ID NO:2) and/or2B (SEQ ID NO:4), with or without the signal sequence(s) and propeptide.

The chimeric polypeptide or hybrid may be administered as part of apharmaceutical composition comprising at least one excipient.

The invention also provides the above-described chimeric and hybridpolypeptides themselves, polynucleotides encoding them, a cultured humanembryonic cells comprising the polynucleotides, and methods of producingsuch chimeric and hybrid polypeptides, and the polypeptides produced bysuch methods.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. Schematic of one type of Factor IX chimeric polypeptide, aFactor IX-Fc hybrid.

FIG. 2. Group mean FIXFc concentration versus time profiles; nominaldose comparison.

FIG. 3. Group mean FIXFc activity versus time profiles; nominal dosecomparison.

FIG. 4. The baseline subtraction decision tree.

FIG. 5. Dose proportional increase in Cmax and AUC for FIX activity.

FIG. 6. Estimated Therapeutic Duration of rFIXFc at 50 (A) and 100 (B)IU/kg.

FIG. 7. Dose proportional increase in Cmax and AUC for FIX antigen.

FIG. 8. Pharmacokinetic estimates for rFIXFc antigen at 50 (A) and 100(B) IU/kg nominal doses.

FIG. 9. Excellent correlation between rFIXFc activity and antigenlevels. Note that due to recalculation of activity PK, as discussed inExample 11, R²=0.946.

FIG. 10. rFIX-Fc domain structure and posttranslational modifications.PRO: Propeptide cleaved by processing enzyme. GLA: contains 12γ-carboxylated glutamic acid (Gla) residues. ACT PEP: activation peptidecleaved to yield active protease. Other modifications: N- andO-glycosylation, Asp(64) β-hydroxylation, Tyr sulfation, Serphosphorylation.

FIG. 11. SDS-PAGE gel of purification intermediates and purified FIXFcmonomer. Samples from different steps in the purification of FIXFc wereanalyzed by non-reducing SDS-PAGE. Lane 1: See Blue Plus MolecularWeight Markers (Invitrogen). Lane 2: empty lane. Lane 3: Protein A load.Lane 4: Protein A eluate. Lane 5: Fractogel DEAE eluate. Lane 6: Q SephFF eluate. Lane 7: final bulk FIXFc. Lane 8: empty lane. Lane 9: finalbulk reduced FIXFc.

FIG. 12. Functional activity of FIXFc in FIX-deficient mice.FIX-deficient mice were dosed intravenously with 219 IU/kg FIXFc (3 or 4per group, 6 groups, n=23) or 200 IU/kg rFIX (3 or 4 per group, 5groups, n=23) at time=0. Blood samples were collected at various timesafter dosing (0.25 hr to 96 hr) and analyzed for clotting activity usingFIX activity assay. * rFIX activity is undetectable in all of the miceat time points later than 48 hr after dosing.

FIG. 13. Whole blood clotting time of FIXFc versus recombinant FIX inFIX-deficient mice. FIX-deficient mice (6 per group) were dosedintravenously with 50 IU/kg FIXFc or 50 IU/kg rFIX. Blood samples werecollected before dosing and at various times after dosing. Blood sampleswere incubated at 37° C. and were visually inspected for the presence ofa blood clot once per minute. The time needed for a clot to form wasrecorded and, once the clotting activity returned to baseline (i.e. noclot formation), no additional samples were obtained (samples collected15 min to 144 hr for FIXFc or 15 min to 72 hr for rFIX).

FIG. 14. Pharmacodynamics of FIXFc in FIX-deficient mice. FIX-deficientmice were dosed with 219 IU/kg FIXFc (5 per group, 6 groups, n=30) or200 IU/kg rFIX (4 or 5 per group, 6 groups, n=28) on Day 0, 4 and 8.Plasma samples were collected by cardiac puncture at 15 min and 96 hrafter each dose and clotting activity was measured using a FIX activityassay. Plasma was also collected by tail bleeds at 8, 24, 48, and 72 hrafter each dose. FIXFc levels were measured in all of the samples usingan ELISA specific for FIXFc. (A) Measured v. Calculated Activity.Clotting activity for FIXFc was measured using FIX activity assay 15 minand 96 h after three doses. The in vitro clotting activity for FIXFc wasdetermined to be 43.8±5.4 IU/mg. Based on this activity (IU/mg) and themeasured protein levels, a calculated plasma clotting activity level wasdetermined for time points at 15 min, 8, 24, 48, 72 and 96 h after eachdose. (B) In FIX-deficient mice treated with up to three doses of 200IU/kg rFIX, FIX levels were measured using FIX-specific ELISA. Using themeasured specific activities of FIXFc and rFIX, it was possible tocompare calculated clotting activity for all samples analyzed by ELISA.

FIG. 15. Pharmacokinetics and pharmacodynamics of FIXFc in FIX-deficientdogs. Two dogs with hemophilia B were intravenously infused with 140IU/kg FIXFc. Blood samples were collected at 5, 15, and 30 min, and at1, 2, 4, 6, 8, 12, 24, 27, 30, 48, 51, 54, 72, 80, 96, 126, 144, and 168hr. (A) A sandwich ELISA utilizing a FIX capture antibody and Fc-HRPdetection antibody was used to measure the concentration of intact FIXFcin the Hemophilic B dog plasma samples. (B) FIX clotting activity wasmeasured for all time points with respect to a standard curve generatedwith FIXFc. (C) Blood collected from animals was immediately analyzedfor whole blood clotting time. Blood samples were incubated at 28° C.and were visually inspected for the presence of a clot once per minute,and the time in which a clot formed was recorded.

FIG. 16. Pharmacokinetics of FIXFc in Cynomolgus monkeys. Monkeys wereadministered a single dose (0.5, 2, and 10 mg/kg, corresponding toapproximately 25, 100 or 500 IU/kg) of FIXFc (n=2, 3, and 3,respectively). Blood samples were collected at 0.25, 0.5, 1, 8, 24, 48,72, 96, 120, 144 and 168 hr post-dose and plasma prepared for analysisof protein concentration by FIXFc-specific ELISA.

FIG. 17. rFIXFc and BENEFIX™ show comparable activity and dose responsein whole blood from HemB mice. (A) ROTEM® Parameters. rFIX or BENEFIX™were spiked into HemB mouse blood and clotting parameters were measuredby ROTEM®. (B)-(D) Dose response, measuring (B) CT, (C) CFT, and (D)Alpha-angle.

FIG. 18. Evaluation of acute efficacy in tail clip bleeding model ofHemophiliac mice.

FIG. 19. (A) Blood loss following tail clip in individual HemB micetreated with rFIXFc or BENEFIX™. (B) Dose response of rFIXFc andBENEFIX™ in median blood loss following tail clip in HemB mice.

FIG. 20. Tail vein transection (TVT) bleeding model of HemB mice: amodel for the venous bleeding characteristic of severe hemophiliapatients.

FIG. 21. Prolonged activity of rFIXFc relative to BENEFIX™ in treatedHemB mice by whole blood ROTEM®. (A) CT, (B) CFT, (C) Alpha-angle, and(D) Partial correlation between whole blood clotting activity (CT) byROTEM® versus plasma activity by aPTT.

FIG. 22. Prolonged efficacy of FIXFc relative to BENEFIX™ in tail veintransection (TVT) bleeding model of HemB mice. (A) Survival: Survivalrates were comparable in mice receiving BENEFIX™24 hours pre TVT as inmice receiving rFIXFc 72 hours pre TVT, and (B) Rebleed: Bleeding rateswere comparable in mice receiving BENEFIX™ 24 hours pre TVT as in micereceiving rFIXFc 72 hours pre TVT.

FIG. 23. Correlation between incremental recovery of rFIXFc activityversus body weight in 12 subjects who received a single dose of 12.5 to100 IU/kg of rFIXFc.

FIG. 24. Monte Carlo simulation using the structural PK model of rFIXFcactivity to construct the activity-time profiles to achieve trough of 1IU/dL above baseline following weekly (A), every 10 days (B), or everytwo week dosing regimens (C). The median population PK parameters andrelevant inter- and intra-subject variabilities were adopted from theclinical Phase1/2a study. 1000 subjects were simulated per dosingregimen with 14 to 16 sampling points for each subject, and the mean±SDof the activity-time profiles of the 1000 subjects was constructedgraphically for different dosing regimens.

FIG. 25. Monte Carlo simulation for rFIXFc doses to achieve trough of 1IU/dL (1%), based on recalculated pharmacokinetic data. (A) once weekly,(B) every 10 days, and (C) every two weeks.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of treating Factor IXdeficiency, e.g., Hemophilia B, with Factor IX using a longer dosinginterval and/or improved pharmacokinetic parameters than is possiblewith currently known Factor IX products. The present invention alsoprovides improved Factor IX chimeric polypeptides, Factor IX chimericpolynucleotides, and methods of production.

“Administering,” as used herein, means to give a pharmaceuticallyacceptable Factor IX polypeptide of the invention to a subject via apharmaceutically acceptable route. Preferred routes of administrationare intravenous, e.g., intravenous injection and intravenous infusion,e.g., via central venous access. Additional routes of administrationinclude subcutaneous, intramuscular, oral, nasal, and pulmonaryadministration, preferably subcutaneous. Factor IX chimeric polypeptidesand hybrid proteins may be administered as part of a pharmaceuticalcomposition comprising at least one excipient. Advantages of the presentinvention include: improved regimen compliance; reduced break throughbleeds; increased protection of joints from bleeds; prevention of jointdamage; reduced morbidity; reduced mortality; prolonged protection frombleeding; decreased thrombotic events; and improved quality of life.

“Chimeric polypeptide,” as used herein, means a polypeptide thatincludes within it at least two polypeptides (or portions thereof suchas subsequences or peptides) from different sources. Chimericpolypeptides may include two, three, four, five, six, seven, or morepolypeptides or portions thereof from different sources, such asdifferent genes, different cDNAs, or different animal or other species.Chimeric polypeptides may include one or more linkers joining thedifferent polypeptides or portions thereof. Thus, the polypeptides orportions thereof may be joined directly or they may be joinedindirectly, via linkers, or both, within a single chimeric polypeptide.Chimeric polypeptides may include additional peptides such as signalsequences and sequences such as 6His and FLAG that aid in proteinpurification or detection. In addition, chimeric polypeptides may haveamino acid or peptide additions to the N- and/or C-termini. Exemplarychimeric polypeptides of the invention are Factor IX-FcRn BP chimericpolypeptides, e.g., Factor IX-Fc chimeric polypeptides such as the FIXFcin FIG. 1, SEQ ID NO:2 (Table 2) and Examples 1-4, with or without itssignal sequence and propeptide. Another exemplary chimeric polypeptidesof the invention include, but are not limited to, Factor IX-XTENchimeric polypeptides. Factor IX can be fused to either N-terminus orC-terminus of XTEN.

The chimeric polypeptide may comprise a sequence at least 90% or atleast 95% or 100% identical to the Factor IX and FcRn BP, e.g., the Fcamino acid sequence shown in Table 2A without a signal sequence andpropeptide sequence (amino acids 1 to 642 of SEQ ID NO:2), oralternatively, with a propeptide sequence, or alternatively with asignal sequence and a propeptide sequence.

“Culture,” “to culture” and “culturing,” as used herein, means toincubate cells under in vitro conditions that allow for cell growth ordivision or to maintain cells in a living state. “Cultured cells,” asused herein, means cells that are propagated in vitro.

“Factor IX” and “FIX,” as used herein, means functional Factor IXpolypeptide in its normal role in coagulation, unless otherwisespecified. Thus, the term Factor IX includes variant polypeptides thatare functional and the polynucleotides that encode such functionalvariant polypeptides. Preferred Factor IX polypeptides are the human,bovine, porcine, canine, feline, and murine Factor IX polypeptides. Thefull length polypeptide and polynucleotide sequences of Factor IX areknown, as are many functional variants, e.g., fragments, mutants andmodified versions. Factor IX polypeptides include full-length Factor IX,full-length Factor IX minus Met at the N-terminus, full-length Factor IXminus the signal sequence, mature Factor IX (minus the signal sequenceand propeptide), and mature Factor IX with an additional Met at theN-terminus. Factor IX is preferably made by recombinant means(“recombinant Factor IX” or “rFIX”), i.e., it is not naturally occurringor derived from plasma.

A great many functional Factor IX variants are known. Internationalpublication number WO 02/040544 A3, which is herein incorporated byreference in its entirety, discloses mutants that exhibit increasedresistance to inhibition by heparin at page 4, lines 9- and page 15,lines 6-31. International publication number WO 03/020764 A2, which isherein incorporated by reference in its entirety, discloses Factor IXmutants with reduced T cell immunogenicity in Tables 2 and 3 (on pages14-24), and at page 12, lines 1-27. International publication number WO2007/149406 A2, which is herein incorporated by reference in itsentirety, discloses functional mutant Factor IX molecules that exhibitincreased protein stability, increased in vivo and in vitro half-life,and increased resistance to proteases at page 4, line 1 to page 19, line11. WO 2007/149406 A2 also discloses chimeric and other variant FactorIX molecules at page 19, line 12 to page 20, line 9. Internationalpublication number WO 08/118,507 A2, which is herein incorporated byreference in its entirety, discloses Factor IX mutants that exhibitincreased clotting activity at page 5, line 14 to page 6, line 5.International publication number WO 09/051,717 A2, which is hereinincorporated by reference in its entirety, discloses Factor IX mutantshaving an increased number of N-linked and/or O-linked glycosylationsites, which results in an increased half-life and/or recovery at page9, line 11 to page 20, line 2. International publication number WO09/137,254 A2, which is herein incorporated by reference in itsentirety, also discloses Factor IX mutants with increased numbers ofglycosylation sites at page 2, paragraph [006] to page 5, paragraph[011] and page 16, paragraph [044] to page 24, paragraph [057].International publication number WO 09/130,198 A2, which is hereinincorporated by reference in its entirety, discloses functional mutantFactor IX molecules that have an increased number of glycosylationsites, which result in an increased half-life, at page 4, line 26 topage 12, line 6. International publication number WO 09/140,015 A2,which is herein incorporated by reference in its entirety, disclosesfunctional Factor IX mutants that an increased number of Cys residues,which may be used for polymer (e.g., PEG) conjugation, at page 11,paragraph [0043] to page 13, paragraph [0053].

In addition, hundreds of non-functional mutations in Factor IX have beenidentified in hemophilia patients, many of which are disclosed in Table1, at pages 11-14 of International publication number WO 09/137,254 A2,which is herein incorporated by reference in its entirety. Suchnon-functional mutations are not included in the invention, but provideadditional guidance for which mutations are more or less likely toresult in a functional Factor IX polypeptide.

The Factor IX (or Factor IX portion of a chimeric polypeptide) may be atleast 90% or at least 95% or 100% identical to a Factor IX amino acidsequence shown in Table 2A without a signal sequence and propeptidesequence (amino acids 1 to 415 of SEQ ID NO:2), or alternatively, with apropeptide sequence, or with a propeptide and signal sequence (fulllength Factor IX).

Factor IX coagulant activity is expresses as International Unit(s) (IU).One IU of Factor IX activity corresponds approximately to the quantityof Factor IX in one milliliter of normal human plasma. Several assaysare available for measuring Factor IX activity, including the one stageclotting assay (activated partial thromboplastin time; aPTT), thrombingeneration time (TGA) and rotational thromboelastometry (ROTEM®). See,e.g., Example 3.

“FcRn binding partner,” or “FcRn BP” as used herein, means functionalneonatal Fc receptor (FcRn) binding partners, unless otherwisespecified. An FcRn binding partner is any molecule that can bespecifically bound by the FcRn receptor with consequent active transportby the FcRn receptor of the FcRn binding partner. Thus, the term FcRn BPincludes any variants of IgG Fc that are functional. For example, theregion of the Fc portion of IgG that binds to the FcRn receptor has beendescribed based on X-ray crystallography (Burmeister et al. 1994, Nature372:379, incorporated herein by reference in its entirety). The majorcontact area of the Fc with the FcRn is near the junction of the CH2 andCH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain.FcRn BPs include whole IgG, the Fc fragment of IgG, and other fragmentsof IgG that include the complete binding region of FcRn. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain. References made to aminoacid numbering of immunoglobulins or immunoglobulin fragments, orregions, are all based on Kabat et al. 1991, Sequences of Proteins ofImmunological Interest, U.S. Department of Public Health, Bethesda; MD,incorporated herein by reference in its entirety. (The FcRn receptor hasbeen isolated from several mammalian species including humans. Thesequences of the human FcRn, rat FcRn, and mouse FcRn are known (Storyet al. 1994, J. Exp. Med. 180: 2377), incorporated herein by referencein its entirety.) An FcRn BP may comprise the CH2 and CH3 domains of animmunoglobulin with or without the hinge region of the immunoglobulin.Exemplary FcRn BP variants are provided in WO 2004/101740 and WO2006/074199, incorporated herein by reference in its entirety.

FcRn BP also include albumin and fragments thereof that bind to theFcRn. Preferably the albumin is human albumin. Factor IX can be fused toeither the N-terminal end of the albumin or to the C-terminal end of thealbumin, provided the Factor IX component of the Factor IX-albuminfusion protein can be processed by an enzymatically-active proproteinconvertase to yield a processed Factor IX-containing polypeptide.Examples of albumin, e.g., fragments thereof, that may be used in thepresent invention are known. e.g., U.S. Pat. No. 7,592,010; U.S. Pat.No. 6,686,179; and Schulte, Thrombosis Res. 124 Suppl. 2:S6-S8 (2009),each of which is incorporated herein by reference in its entirety.

FcRn BP (or FcRn BP portion of a chimeric polypeptide) may contain oneor more mutations, and combinations of mutations.

FcRn BP (or FcRn BP portion of a chimeric polypeptide) may containmutations conferring increased half-life such as M252Y, S254T, T256E,and combinations thereof, as disclosed in Oganesyan et al., Mol.Immunol. 46:1750 (2009), which is incorporated herein by reference inits entirety; H433K, N434F, and combinations thereof, as disclosed inVaccaro et al., Nat. Biotechnol. 23:1283 (2005), which is incorporatedherein by reference in its entirety; the mutants disclosed at pages 1-2,paragraph [0012], and Examples 9 and 10 of U.S. 2009/0264627 A1, whichis incorporated herein by reference in its entirety; and the mutantsdisclosed at page 2, paragraphs [0014] to [0021] of U.S. 20090163699 A1,which is incorporated herein by reference in its entirety.

FcRn BP (or FcRn BP portion of a chimeric polypeptide) may also includethe following mutations: The Fc region of IgG can be modified accordingto well recognized procedures such as site directed mutagenesis and thelike to yield modified IgG or Fc fragments or portions thereof that willbe bound by FcRn. Such modifications include modifications remote fromthe FcRn contact sites as well as modifications within the contact sitesthat preserve or even enhance binding to the FcRn. For example thefollowing single amino acid residues in human IgG1 Fc (Fcy1) can besubstituted without significant loss of Fc binding affinity for FcRn:P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A,S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A,E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F,N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A,N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q,A330S, P331A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A,R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A,Y373A, S375A D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A,N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A,Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A,S440A, S444A, and K447A, where for example P238A represents wild typeproline substituted by alanine at position number 238. In addition toalanine other amino acids may be substituted for the wild type aminoacids at the positions specified above. Mutations may be introducedsingly into Fc giving rise to more than one hundred FcRn bindingpartners distinct from native Fc. Additionally, combinations of two,three, or more of these individual mutations may be introduced together,giving rise to hundreds more FcRn binding partners. Certain of thesemutations may confer new functionality upon the FcRn binding partner.For example, one embodiment incorporates N297A, removing a highlyconserved N-glycosylation site. The effect of this mutation is to reduceimmunogenicity, thereby enhancing circulating half-life of the FcRnbinding partner, and to render the FcRn binding partner incapable ofbinding to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA, without compromisingaffinity for FcRn (Routledge et al. 1995, Transplantation 60:847, whichis incorporated herein by reference in its entirety; Friend et al. 1999,Transplantation 68:1632, which is incorporated herein by reference inits entirety; Shields et al. 1995, J. Biol. Chem. 276:6591, which isincorporated herein by reference in its entirety). Additionally, atleast three human Fc gamma receptors appear to recognize a binding siteon IgG within the lower hinge region, generally amino acids 234-237.Therefore, another example of new functionality and potential decreasedimmunogenicity may arise from mutations of this region, as for exampleby replacing amino acids 233-236 of human IgG1 “ELLG” to thecorresponding sequence from IgG2 “PVA” (with one amino acid deletion).It has been shown that FcγRI, FcγRII, and FcγRIII which mediate variouseffector functions will not bind to IgG1 when such mutations have beenintroduced (Ward and Ghetie 1995, Therapeutic Immunology 2:77, which isincorporated herein by reference in its entirety; and Armour et al.1999, Eur. J. Immunol. 29:2613, which is incorporated herein byreference in its entirety). As a further example of new functionalityarising from mutations described above, affinity for FcRn may beincreased beyond that of wild type in some instances. This increasedaffinity may reflect an increased “on” rate, a decreased “off” rate orboth an increased “on” rate and a decreased “off” rate. Mutationsbelieved to impart an increased affinity for FcRn include T256A, T307A,E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591, which isincorporated herein by reference in its entirety).

The FcRn BP (or FcRn BP portion of a chimeric polypeptide) may be atleast 90% or at least 95% or 100% identical to the Fc amino acidsequence shown in Table 2A or B without a signal sequence (amino acids 1to 227 of SEQ ID NO:2), or alternatively, with a signal sequence.

“Hybrid” polypeptides and proteins, as used herein, means a combinationof a chimeric polypeptide with a second polypeptide. The chimericpolypeptide and the second polypeptide in a hybrid may be associatedwith each other via non-covalent protein-protein interactions, such ascharge-charge or hydrophobic interactions. The chimeric polypeptide andthe second polypeptide in a hybrid may be associated with each other viacovalent bond(s) such as disulfide bonds. The chimeric peptide and thesecond peptide may be associated with each other via more than one typeof bond, such as non-covalent and disulfide bonds. Hybrids are describedin WO 2004/101740, WO2005/001025, U.S. Pat. No. 7,404,956, U.S. Pat. No.7,348,004, and WO 2006/074199, each of which is incorporated herein byreference in its entirety. The second polypeptide may be a second copyof the same chimeric polypeptide or it may be a non-identical chimericpolypeptide. In preferred embodiments, the second polypeptide is apolypeptide comprising an FcRn BP, e.g., Fc. In preferred embodiments,the chimeric polypeptide is a Factor IX-FcRn BP, e.g., Factor IX-Fcchimeric polypeptide, and the second polypeptide consists essentially ofFc. See, e.g., FIG. 1, Examples 1-3, and Table 2 (SEQ ID NOs:2 and 4).See, e.g., U.S. Pat. No. 7,404,956, which is incorporated herein byreference. in its entirety.

The second polypeptide in a hybrid may comprise or consist essentiallyof a sequence at least 90% or at least 95% or 100% identical to theamino acid sequence shown in Table 2B without a signal sequence (aminoacids 1 to 227 of SEQ ID NO:4), or alternatively, with a signalsequence.

The polypeptide of the present invention also includes Factor IX fusedto one or more XTEN polypeptides. Schellenburger et al., Nat. Biotech.27:1186-90 (2009), which is incorporated herein by reference in itsentirety. Factor IX can be fused to either the N-terminal end of theXTEN polypeptide or to the C-terminal end of the XTEN polypeptide. XTENpolypeptides include, but not limited to, those disclosed in WO2009/023270, WO 2010/091122, WO 2007/103515, US 2010/0189682, and US2009/0092582, each of which is incorporated herein by reference in itsentirety.

“Dosing interval,” as used herein, means the amount of time that elapsesbetween multiple doses being administered to a subject. The dosinginterval in the methods of the invention using a chimeric FIX-FcRn BP,e.g., a chimeric FIX-Fc, may be at least about one and one-half to eighttimes longer than the dosing interval required for an equivalent amount(in IU/kg) of said Factor IX without the FcRn BP, e.g., Fc portion(i.e., a polypeptide consisting of said FIX). The dosing interval whenadministering, e.g., a Factor IX-Fc chimeric polypeptide (or a hybrid)of the invention may be at least about one and one-half times longerthan the dosing interval required for an equivalent amount of saidFactor IX without the FcRn BP, e.g., Fc, portion (i.e., a polypeptideconsisting of said Factor IX). The dosing interval may be at least aboutone and one-half to eight times longer than the dosing interval requiredfor an equivalent amount of said Factor IX without, e.g., the Fc portion(or a polypeptide consisting of said Factor IX).

In some embodiments, the dosing interval is 6-18, 6-10, 9-18, at least6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, or at least 18 days. The dosing interval may be at least about onceweekly, and may be 6-10 days, e.g., about 7-10, about 7-9, about 7-8,about 8-10, about 9-10, about 6-7, about 8-9, about 6, about 7, about 8,about 9, or about 10 days.

The dosing interval may be 9-18 days, e.g., about 9-17, about 9-16,about 9-15, about 9-14, about 9-13, about 9-12, about 9-11, about 9-10days, about 10-18, about 11-18, about 12-18, about 13-18, about 14-18,about 15-18, about 16-18, about 17-18 days, about 10-11, about 11-12,about 12-13, about 13-14, about 14-15, about 15-16, and about 16-17days, about 9, about 10, about 11, about 12, about 13, about 14, about15, about 16, about 17, or about 18 days. The dosing interval may beabout 10-14 days. The dosing interval may be about every two weeks ortwice monthly. The dosing interval may be longer than 18 days, e.g.,about 19, about 20, about 21, about 22, about 23, about 24, about 25,about 26, about 27, about 28, about 29, about 30, about 31, about 32,about 33, about 34, about 35, about 36, about 37, about 38, about 39, orabout 40 days. The dosing interval may be a fixed interval, e.g., 7 daysfor 25-50 IU/kg, 10-13 days for 50-100 IU/kg, or 14 days for 100-150IU/kg. The fixed interval and dose are determined such that thecombination of interval and dose will result in a trough of at leastabout 1-5 or at least about 1-3, or at least about 1, at least about 2,or at least about 3 IU/dl FIX activity in a population of subjects or inan individual subject. The fixed dosing interval may also be 7 days for20-50 IU/kg, 10-14 days for 50-100 IU/kg, 14-16 days for 100-150 IU/kg,7 days for 10-50 IU/kg, 10-13 days for 15-100 IU/kg, or 14-15 days for50-150 IU/kg. The fixed dosing interval may also be 7 days for 10-30IU/kg, 10 days 15-50 IU/kg, 11 days 20-70 IU/kg, 12 days 25-85 IU/kg, 13days 30 to 100 IU/kg, 14 days 40 to 125 IU/kg, and 15 days for 50-150IU/kg.

In preferred embodiments, the dosing interval is 20 IU/kg once weekly,40 IU/kg every 10 days, or 100 IU/kg every two weeks (twice monthly).

The dosing interval may, alternatively, be an individualized intervalthat is determined for each subject based on pharmacokinetic data orother information about that subject. The individualized dose/dosinginterval combination may be the same as those for fixed intervalregimens in the preceding paragraphs, or may differ, as illustrated inthe Examples. The regimen may initially be at a fixed dosing interval,and then it may change to an individualized dosing interval.

“On-demand treatment,” as used herein, means treatment that is intendedto take place over a short course of time and is in response to anexisting condition, such as a bleeding episode, or a perceived shortterm need such as planned surgery. Conditions that may require on-demandtreatment include a bleeding episode, hemarthrosis, muscle bleed, oralbleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma,trauma capitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, or bleeding in the illiopsoassheath. Bleeding episodes other than these are also included. Thesubject may be in need of surgical prophylaxis, peri-operativemanagement, or treatment for surgery. Such surgeries include minorsurgery, major surgery, tooth extraction, tonsillectomy, otherdental/thoraco-facial surgeries, inguinal herniotomy, synovectomy, totalknee replacement, other joint replacement, craniotomy, osteosynthesis,trauma surgery, intracranial surgery, intra-abdominal surgery,intrathoracic surgery. Surgeries other than these are also included.Additional conditions that may require on-demand treatment include thoselisted in Table 26.

Additional conditions that may require on-demand treatment include minorhemorrhage, hemarthroses, superficial muscle hemorrhage, soft tissuehemorrhage, moderate hemorrhage, intramuscle or soft tissue hemorrhagewith dissection, mucous membrane hemorrhage, hematuria, majorhemorrhage, hemorrhage of the pharynx, hemorrhage of the retropharynx,hemorrhage of the retroperitonium, hemorrhage of the central nervoussystem, bruises, cuts, scrapes, joint hemorrhage, nose bleed, mouthbleed, gum bleed, intracranial bleeding, intraperitoneal bleeding, minorspontaneous hemorrhage, bleeding after major trauma, moderate skinbruising, or spontaneous hemorrhage into joints, muscles, internalorgans or the brain. Additional reasons for on-demand treatment includethe need for peri-operative management for surgery or dental extraction,major surgery, extensive oral surgery, urologic surgery, hernia surgery,orthopedic surgery such as replacement of knee, hip, or other majorjoint.

ABBREVIATIONS

-   AUC_(INF) Area under the concentration-time curve from zero to    infinity-   AUC_(α) Area under the concentration-time curve over the    distribution phase-   AUC_(β) Area under the concentration-time curve over the elimination    phase-   Alpha HL Distribution phase half-life-   Beta HL Elimination phase half-life; also referred to as t_(1/2)-   C168 Estimated FIXFc activity above baseline at approximately 168 h    after dose-   C_(max) Maximum concentration, occurring at T_(max)-   CV % Percent coefficient of variation-   Cl Clearance-   IVR in vivo recovery (%)-   K-Value Incremental recovery-   MRT Mean residence time-   N Number-   NC Not Calculable-   NR Not Reported-   SD Standard Deviation-   SE Standard Error-   TBLP1 Model-predicted time after dose when FIXFc activity has    declined to approximately 1 IU/dL above baseline-   TBLP3 Model-predicted time after dose when FIXFc activity has    declined to approximately 3 IU/dL above baseline-   TBLP5 Model-predicted time after dose when FIXFc activity has    declined to approximately 5 IU/dL above baseline-   V_(SS) Volume of distribution at steady state-   V₁ Volume of distribution of the central compartment

Pharmacokinetic (PK) parameters include the terms above and thefollowing terms, which have their ordinary meaning in the art, unlessotherwise indicated. Some of the terms are explained in more detail inthe Examples. PK parameters may be based on FIX antigen level (oftendenoted parenthetically herein as “antigen”) or FIX activity level(often denoted parenthetically herein as “activity”). In the literature,PK parameters are often based on FIX activity level due to the presencein the plasma of some patients of endogenous, inactive FIX, whichinterferes with the ability to measure administered (i.e., exogenous)FIX using antibody against FIX. However, when FIX is administered aspart of a fusion protein containing a heterologous polypeptide such as aFcRn BP, administered (i.e., exogenous) FIX antigen may be accuratelymeasured using antibody to the heterologous polypeptide. In addition,certain PK parameters may be based on model predicted data (oftendenoted parenthetically herein as “model predicted”) or on observed data(often denoted parenthetically herein as “observed”), and preferably arebased on observed data.

“Baseline,” as used herein, is the lowest measured plasma Factor IXlevel in a subject prior to administering a dose. In the first-in-humanstudy described in Example 1, the Factor IX plasma levels were measuredat two time points prior to dosing: at a screening visit and immediatelyprior to dosing. Predose times were treated as zero (baseline) for thepurpose of calculations, i.e., to generate “baseline subtracted” data.See, e.g., FIG. 4. Alternatively, (a) the baseline in patients whosepretreatment FIX activity is <1%, who have no detectable FIX antigen,and have nonsense genotypes is defined as 0%, (b) the baseline forpatients with pretreatment FIX activity <1% and who have detectable FIXantigen is set at 0.5%, (c) the baseline for patients whose pretreatmentFIX activity is between 1-2% is Cmin (the lowest activity throughout thePK study), and (d) the baseline for patients whose pretreatment FIXactivity is ≧2% is 2%. Activity above the baseline pre-dosing isconsidered residue drug from prior treatment, and was decayed tobaseline and subtracted from the PK data following rFIXFc dosing. SeeExample 11.

“Area under the plasma concentration versus time curve” (“AUC”), which,as used herein, is based upon the rate and extent of elimination ofFactor IX following administration. AUC is determined over a specifiedtime period, such as 12, 18, 24, 36, 48, or 72 hours, or for infinityusing extrapolation based on the slope of the curve. Unless otherwisespecified herein, AUC is determined for infinity (AUC_(INF)). AUC mayalso be calculated on a per dose basis. As with many of the other PKparameters, the determination of AUC may be carried out in a singlesubject, or in a population of subjects for which the average iscalculated. In Example 1, the mean AUC/dose in the patient populationwas 32.44 IU*h/dL per IU/kg and the range for individual subjects was21.80-54.30 IU*h/dL per IU/kg. (See Table 13 for mean AUC/dose based onactivity.) Therefore, the mean AUC/dose in a patient population may beabout 26-40, about 26, about 27, about 28, about 29, about 30, about 31,about 32, about 33, about 34, about 35, about 36, about 37, about 38,about 39, or about 40 IU*h/dL per IU/kg. See Table 14 for AUC/dose andother AUC parameters based on antigen.

“In vivo recovery” (“IVR”) is represented by the incremental recovery(K-value), which is the observed peak activity minus predose level andthen divided by the dose. IVR may also be calculated on a percentagebasis, as is described in the Examples. For clarity, the units (K valueor IU/dl per IU/kg versus %) are used herein. The mean IVR can bedetermined in a patient population, or the individual IVR can bedetermined in a single subject. The FIXFc used in the first-in-humanstudy described in Example 1 exhibited a mean IVR of about 0.93 IU/dlper IU/kg in the patient population; and an IVR in each subject thatranged from 0.62 to 1.17 IU/dl per IU/kg (Table 13). Therefore, thechimeric polypeptide of the invention exhibits an mean IVR in a patientpopulation of 0.85-1.15 (e.g., about 0.85, about 0.86, about 0.87, about0.88, about 0.89, about 0.90, about 0.91, about 0.92, about 0.93, about0.94, about 0.95, about 0.96, about 0.97, about 0.98, about 0.99, about1.0, about 1.05, about 1.10, about 1.15) and an IVR in a subject of atleast about 0.6, about 0.7, 0.8, about 0.9, about 1.0, about 1.1, orabout 1.2 IU/dl per IU/kg.

“Clearance rate” (“CL”), as used herein, is a measure of the body'sability to eliminate a drug, and is expressed as the volume of plasmacleared of drug over time. The FIXFc used in the study described inExample 1 exhibited a mean CL of about 3.36 ml/hour/kg (see Table 13),which is about 2.5 fold lower than the CL (8.2 ml/hour/kg) of apolypeptide consisting of Factor IX (BENEFIX™); the range of CL valuesin individual subjects was 1.84-4.58 ml/h/kg. Therefore, a chimericpolypeptide of the invention exhibits a mean CL in a population of3.0-3.72, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, or 3.72 mL/hour/kg ForCL based on antigen, see Table 14.

“Mean residence time” (“MRT”), as used herein, is a measure of theaverage lifetime of drug molecules in the body. The FIXFc used in thestudy described in Example 1 exhibited a mean MRT of about 68.05 hours(see Table 13); the range of MRT values was 53.1-85.8 hours inindividual subjects. Therefore, a chimeric polypeptide of the inventionexhibits a mean MRT in a population of 60-78, about 60, about 62, about64, about 66, about 68, about 70, about 72, about 74, about 76, or about78 hours and a MRT in a subject of at least about 50, about 55, about60, about 65, about 70, about 75, about 80, about 85, or about 90 hours.For MRT based on antigen, see Table 14.

“t_(1/2β)” or t_(1/2 beta)” or “Beta HL,” as used herein, is half-lifeassociated with elimination phase, t_(1/2β)=(ln 2)/elimination rateconstant associated with the terminal phase. In the study described inExample 1, the FIXFc used exhibited a mean t_(1/2z) in a patientpopulation that was about 52.5 hours (see Table 13) and the range oft_(1/2 β) values in individual subjects was 47-60 hours. Therefore, achimeric polypeptide of the invention exhibits an average t_(1/2β)greater than about 47, about 48, about 49, about 50, about 51, about 52,about 53, about 54, about 55, about 56, about 57, about 58, about 59, orabout 60 hours. For t_(1/2β) based on antigen, see Table 14.

“Trough,” as used herein, is the lowest plasma Factor IX activity levelreached after administering a dose of chimeric polypeptide of theinvention or another Factor IX molecule and before the next dose isadministered, if any. Trough is used interchangeably herein with“threshold.” Baseline Factor IX levels are subtracted from measuredFactor IX levels to calculate the trough level. In some embodiments, thetrough is 1-5 or 1-3 IU/dl after about 6, about 7, about 8, about 9,about 10, about 11, about 12, about 13 or about 14 days. In someembodiments, the plasma level of the chimeric polypeptide reaches anaverage trough of at least about 1 IU/dl after at least about 6 days inat least about 70%, at least about 80%, at least about 90%, or about100% of a patient population or reaches a trough of at least about 1, 2,3, 4, or 5 IU/dl after at least about 6 days in a subject. In someembodiments, the plasma level of said chimeric polypeptide reaches anaverage trough of about 1-5 or 1-3 IU/dl. Such trough or average troughmay be reached after about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, or about 40 days.

“Volume of distribution at steady state (Vss),” as used herein, is theapparent space (volume) into which a drug distributes. Vss=the amount ofdrug in the body divided by the plasma concentration at steady state. InExample 1, the mean Vss found in the population was about 226 mL/kg andthe range for subjects was about 145-365 mL/kg. (See Table 13.) Thus,the mean Vss in a patient population may be 200-300, about 200, about210, about 220, about 230, about 240, about 250, about 260, about 270,about 280, about 290, or about 300 mL/kg. The Vss for individualsubjects may be about 145, about 150, about 160, about 170, about 180,about 190, about 200, about 210, about 220, about 230, about 240, about250, about 260, about 270, about 280, about 290, about 300, about 310,about 320, about 330, about 340, about 350, about 360, or about 370ml/kg. For Vss based on antigen, see Table 14.

“Polypeptide,” “peptide” and “protein” are used interchangeably andrefer to a polymeric compound comprised of covalently linked amino acidresidues.

“Polynucleotide” and “nucleic acid” are used interchangeably and referto a polymeric compound comprised of covalently linked nucleotideresidues. Polynucleotides may be DNA, cDNA, RNA, single stranded, ordouble stranded, vectors, plasmids, phage, or viruses. Polynucleotidesinclude those in Table 1, which encode the polypeptides of Table 2 (seeTable 1). Polynucleotides also include fragments of the polynucleotidesof Table 1, e.g., those that encode fragments of the polypeptides ofTable 2, such as the Factor IX, Fc, signal sequence, propeptide, 6Hisand other fragments of the polypeptides of Table 2.

“Prophylactic treatment,” as used herein, means administering a FactorIX polypeptide in multiple doses to a subject over a course of time toincrease the level of Factor IX activity in a subject's plasma.Preferably, the increased level is sufficient to decrease the incidenceof spontaneous bleeding or to prevent bleeding in the event of anunforeseen injury. Prophylactic treatment decreases or prevents bleedingepisodes, for example, those described under on-demand treatment.Prophylactic treatment may be fixed or may be individualized, asdiscussed under “dosing interval”, e.g., to compensate for inter-patientvariability.

“Subject,” as used herein means a human or a non-human mammal. Non-humanmammals include mice, dogs, primates, monkeys, cats, horses, cows, pigs,and other domestic animals and small animals. Subjects also includepediatric humans. Pediatric human subjects are birth to 20 years,preferably birth to 18 years, birth to 16 years, birth to 15 years,birth to 12 years, birth to 11 years, birth to 6 years, birth to 5years, birth to 2 years, and 2 to 11 years of age.

The methods of the invention may be practiced on a subject in need ofcontrol or prevention of bleeding or bleeding episodes. Such subjectsinclude those in need of control or prevention of bleeding in minorhemorrhage, hemarthroses, superficial muscle hemorrhage, soft tissuehemorrhage, moderate hemorrhage, intramuscle or soft tissue hemorrhagewith dissection, mucous membrane hemorrhage, hematuria, majorhemorrhage, hemorrhage of the pharynx, hemorrhage of the retropharynx,hemorrhage of the retroperitonium, hemorrhage of the central nervoussystem, bruises, cuts, scrapes, joint hemorrhage, nose bleed, mouthbleed, gum bleed, intracranial bleeding, intraperitoneal bleeding, minorspontaneous hemorrhage, bleeding after major trauma, moderate skinbruising, or spontaneous hemorrhage into joints, muscles, internalorgans or the brain. Such subjects also include those need ofperi-operative management, such as management of bleeding associatedwith surgery or dental extraction.

“Therapeutic dose,” as used herein, means a dose that achieves atherapeutic goal, as described herein. The calculation of the requireddosage of plasma derived Factor IX (pdFIX) is based upon the empiricalfinding that, on average, 1 IU of pdFIX per kg body weight raises theplasma Factor IX activity by approximately 1 IU/dL (1%). On that basis,the required dosage is determined using the following formula:Required units=body weight (kg)×desired Factor IX rise (IU/dL or % ofnormal)×1 (IU/kg per IU/dL)

Because FIXFc, e.g., as described in the Examples and in FIG. 1, has anincremental recovery similar to pdFIX (different from that of BENEFIX™),the required dose is determined using the formula above, or adjusting itslightly. See also Table 26 for specific recommended doses for variouson-demand treatment needs. For pediatric subjects using pdFIX, dosageguidance is the same as for adults. However, pediatric patients may havea lower incremental recovery, and the dosage may therefore need to beadjusted upwards.

The therapeutic doses that may be used in the methods of the inventionare 10-180, 20-180, or 25-180 IU/kg, more specifically, preferred dosesfor a 6-10 day dosing interval are as follows: about 25-110, about30-110, about 40-110, about 50-110, about 60-110, about 70-110, about80-110, about 90-110, and about 100-110; about 30-100, about 30-90,about 30-80, about 30-70, about 30-60, about 30-50, about 30-40 IU/kg;about 40-110, about 50-100, about 60-90, and about 70-80 IU/kg; about40-50, about 50-60, about 60-70, about 70-80, about 80-90, about 90-100,and about 100-110 IU/kg; about 25, about 30, about 35, about 40, about45, about 50, about 55, about 60, about 65, about 70, about 75, about80, about 85, about 90, about 95, about 100, about 105, and about 110IU/kg. A 6-10 day dosing interval includes a weekly dosing interval.Additional therapeutic doses for a 6-10 day, e.g., weekly, dosinginterval include 20-50, 20-100, and 20-180 IU/kg, more specifically,preferred doses for a 6-10 day, e.g., weekly, dosing interval are asfollows: about 20-110, about 20-100, about 20-90, about 20-80, about20-70, about 20-60, about 20-50, about 20-40, about 20-30, about 20-40,and about 20 IU/kg. See also Examples 10 and 11. Doses may be lower than20 IU/kg if effective for a given patient, e.g., about 10, about 11,about 12, about 13, about 14, about 15, about 16, about 17, about 18, orabout 19 IU/kg.

Preferred therapeutic doses for a 9-18 day, e.g., two times monthly,dosing interval are as follows: about 50-180, about 60-180, about70-180, about 80-180, about 90-180, about 100-180, about 110-180, about120-180, about 130-180, about 140-180, about 150-180, about 160-180, andabout 170-180 IU/kg; about 90-170, about 90-160, about 90-150, about90-140, about 90-130, about 90-120, about 90-110, and about 90-100IU/kg; about 100-170, about 110-160, about 120-150, and about 130-140IU/kg; about 90-100, about 100-110, about 110-120, about 120-130, about130-140, about 140-150, about 150-160, and about 160-170 IU/kg; about60, about 70, about 80, about 90, about 95, about 100, about 105, about110, about 115, about 120, about 125, about 130, about 135, about 140,about 145, about 150, about 155, about 160, about 165, about 170, about175, and about 180 IU/kg. See also Examples 10 and 11.

Preferred therapeutic doses are 10-50, 15-100, 20-100, 20-50, 50-100,10, 20, 40, 50, and 100 IU/kg.

The therapeutic dose may be about 20-50, about 20-100, about 20-180,25-110, about 30-110, about 40-110, about 50-110, about 60-110, about70-110, about 80-110, about 90-110, about 100-110, about 30-100, about30-90, about 30-80, about 30-70, about 30-60, about 30-50, about 30-40IU/kg, about 40-110, about 50-100, about 60-90, about 70-80 IU/kg, about40-50, about 50-60, about 60-70, about 70-80, about 80-90, about 90-100,about 100-110 IU/kg, about 20, about 25, about 30, about 35, about 40,about 45, about 50, about 55, about 60, about 65, about 70, about 75,about 80, about 85, about 90, about 95, about 100, about 105, and about110 IU/kg. Such doses are preferred for dosing intervals of about 6-10,about 7-10, about 7-9, about 7-8, about 8-10, about 9-10, about 6-7,about 8-9, about 6, about 7, about 8, about 9, and about 10 days, andonce weekly.

The therapeutic dose may about 90-180, about 100-180, about 110-180,about 120-180, about 130-180, about 140-180, about 150-180, about160-180, and about 170-180 IU/kg. The dose may be about 90-170, about90-160, about 90-150, about 90-140, about 90-130, about 90-120, about90-110, and about 90-100 IU/kg. The dose may be about 100-170, about110-160, about 120-150, and about 130-140 IU/kg. The dose may be about90-100, about 100-110, about 110-120, about 120-130, about 130-140,about 140-150, about 150-160, and about 160-170 IU/kg. The dose may beabout 90, about 95, about 100, about 105, about 110, about 115, about120, about 125, about 130, about 135, about 140, about 145, about 150,about 155, about 160, about 165, about 170, about 175, and about 180IU/kg. Such doses are preferred for dosing interval of about 9-18, about9-17, about 9-16, about 9-15, about 9-14, about 9-13, about 9-12, about9-11, about 9-10, about 10-18, about 11-18, about 12-18, about 13-18,about 14-18, about 15-18, about 16-18, about 17-18, about 10-11, about11-12, about 12-13, about 13-14, about 14-15, about 15-16, and about16-17 days, about 9, about 10, about 11, about 12, about 13, about 14,about 15, about 16, about 17, and about 18 days, one time monthly andtwo times monthly (every two weeks).

Preferred therapeutic dose and dosing intervals are as follows: 20 IU/kgonce weekly, 40 IU/kg every 10 days, and 100 IU/kg every two weeks(twice monthly). Additional combinations of dose and dose intervalinclude: a dose at least about 50 IU/kg and a dosing interval at leastabout 7 days, a dose at least about 100 IU/kg and a dosing interval atleast about 9 days, a dose at least about 100 IU/kg and a dosinginterval at least about 12 days, a dose at least about 150 IU/kg and adosing interval at least about 14 days, 20-50 or 20-100 IU/kg and saiddosing interval is one time weekly, a dose of 20-50 IU/kg and a dosinginterval of 7 days, a dose of 50-100 IU/kg and a dosing interval of10-14 days, or a dose of 100-150 IU/kg and a dosing interval of 14-16days. Preferred combinations of dosing interval and dose also include10-50 IU/kg for 7 days, 15-100 IU/kg for 10-13 days, 50-150 IU/kg for14-15 days, 10-30 IU/kg for 7 days, 15-50 IU/kg for 10 days, 20-70 IU/kgfor 11 days, 25-85 IU/kg for 12 days, 30 to 100 IU/kg for 13 days, 40 to125 IU/kg for 14 days, and 50-150 IU/kg for 15 days.

“Variant,” as used herein, refers to a polynucleotide or polypeptidediffering from the original polynucleotide or polypeptide, but retainingessential properties thereof, e.g., Factor IX coagulant activity or Fc(FcRn binding) activity. Generally, variants are overall closelysimilar, and, in many regions, identical to the original polynucleotideor polypeptide. Variants include polypeptide and polynucleotidefragments, deletions, insertions, and modified versions of originalpolypeptides.

Variant polynucleotides may comprise, or alternatively consist of, anucleotide sequence which is at least 85%, 90%, 95%, 96%, 97%, 98% or99% identical to, for example, the nucleotide coding sequence in SEQ IDNO:1 or 3 (the Factor IX portion, the Fc portion, individually ortogether) or the complementary strand thereto, the nucleotide codingsequence of known mutant and recombinant Factor IX or Fc such as thosedisclosed in the publications and patents cited herein or thecomplementary strand thereto, a nucleotide sequence encoding thepolypeptide of SEQ ID NO:2 or 4 (the Factor IX portion, the Fc portion,individually or together), and/or polynucleotide fragments of any ofthese nucleic acid molecules (e.g., those fragments described herein).Polynucleotides which hybridize to these nucleic acid molecules understringent hybridization conditions or lower stringency conditions arealso included as variants, as are polypeptides encoded by thesepolynucleotides as long as they are functional.

Variant polypeptides may comprise, or alternatively consist of, an aminoacid sequence which is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to, for example, the polypeptide sequence shown in SEQ ID NO:2or 4 (the Factor IX portion, the Fc portion, individually or together),and/or polypeptide fragments of any of these polypeptides (e.g., thosefragments described herein).

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence, it is intended thatthe nucleotide sequence of the nucleic acid is identical to thereference sequence except that the nucleotide sequence may include up tofive point mutations per each 100 nucleotides of the referencenucleotide sequence. In other words, to obtain a nucleic acid having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may be,for example, the entire sequence shown in SEQ ID NO:1 or 3, the ORF(open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toa nucleotide sequence or polypeptide of the present invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(reference or original sequence) and a subject sequence, also referredto as a global sequence alignment, can be determined using the FASTDBcomputer program based on the algorithm of Brutlag et al. (Comp. App.Biosci. (1990) 6:237-245), which is herein incorporated by reference inits entirety In a sequence alignment the query and subject sequences areboth DNA sequences. An RNA sequence can be compared by converting U's toT's. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a FASTDB alignment of DNAsequences to calculate percent identity are: Matrix=Unitary, k-tuple=4,Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0,Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 orthe length of the subject nucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequences of SEQ ID NO:2 (the Factor IX portion, the Fcportion, individually or together) or 4, or a known Factor IX or Fcpolypeptide sequence, can be determined conventionally using knowncomputer programs. A preferred method for determining the best overallmatch between a query sequence (reference or original sequence) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al., Comp. App. Biosci. 6:237-245 (1990), incorporatedherein by reference in its entirety. In a sequence alignment the queryand subject sequences are either both nucleotide sequences or both aminoacid sequences. The result of said global sequence alignment is inpercent identity. Preferred parameters used in a FASTDB amino acidalignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, JoiningPenalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The polynucleotide variants may contain alterations in the codingregions, non-coding regions, or both. Especially preferred arepolynucleotide variants containing alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. Nucleotide variants producedby silent substitutions due to the degeneracy of the genetic code arepreferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.Polynucleotide variants can be produced for a variety of reasons, e.g.,to optimize codon expression for a particular host (change codons in thehuman mRNA to those preferred by a bacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides. For instance, one or more aminoacids can be deleted from the N-terminus or C-terminus of the secretedprotein without substantial loss of biological function. The authors ofRon et al., J. Biol. Chem. 268: 2984-2988 (1993), incorporated herein byreference in its entirety, reported variant KGF proteins having heparinbinding activity even after deleting 3, 8, or 27 amino-terminal aminoacid residues. Similarly, Interferon gamma exhibited up to ten timeshigher activity after deleting 8-10 amino acid residues from the carboxyterminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216(1988), incorporated herein by reference in its entirety.)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993),incorporated herein by reference in its entirety) conducted extensivemutational analysis of human cytokine IL-1a. They used randommutagenesis to generate over 3,500 individual IL-1a mutants thataveraged 2.5 amino acid changes per variant over the entire length ofthe molecule. Multiple mutations were examined at every possible aminoacid position. The investigators found that “[m]ost of the moleculecould be altered with little effect on either [binding or biologicalactivity].” (See Abstract.) In fact, only 23 unique amino acidsequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity from wildtype.

As stated above, polypeptide variants include modified polypeptides.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

The term “about” is used herein to mean approximately, roughly, around,or in the regions of. When the term “about” is used in conjunction witha numerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10 percent, up or down (higher or lower).

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention. All patents and publicationsreferred to herein are expressly incorporated by reference.

EXAMPLE 1 First-In-Human (FiH) Trial

The first-in-human study was an open label, dose-escalation, Phase 1/2study to determine the safety, tolerability and pharmacokinetic (PK)parameters of FIXFc (recombinant human coagulation factor IX fusionprotein). FIXFc is a recombinant fusion protein comprising humanclotting factor IX coupled to the Fc domain from human IgG1. The fusionprotein is expressed in human embryonic kidney cells (HEK 293). SeeExample 3.

FIXFc is being developed for the control and prevention of hemorrhagicepisodes in patients with hemophilia B (congenital factor IX deficiencyor Christmas disease), including the control and prevention of bleedingin surgical settings.

FIXFc is a recombinant fusion protein comprised of coagulation Factor IX(FIX) and an Fc domain of a human antibody (IgG1 isotype). The FIXFcmolecule is heterodimeric with a FIXFc single chain (FIXFc-sc) and an Fcsingle chain (Fc-sc) bound together through two disulfide bonds in thehinge region of Fc. See FIG. 1 and Table 2.

rFIXFc drug product is a clear colorless solution intended forintravenous (IV) administration. rFIXFc is supplied as 1000 IU per a 5mL volume in a 10 mL single use only vial. The Drug Product is packagedin USP Type I glass vials with bromobutyl stoppers and tear-off plainaluminum overseals. rFIXFc drug product contains 200 IU/mL in 10 mMsodium phosphate buffer pH 7.0 with addition of 145 mM NaCl and 0.1%polysorbate 20. The rFIXFc solution should not be diluted.

Study Design. A total of 14 previously treated patients with severehemophilia B were enrolled and treated with FIXFc as an intravenous (IV)infusion over approximately 10 minutes. Six dose levels, 1, 5, 12.5, 25,50, and 100 IU/kg were evaluated in the study. One patient per doselevel was enrolled at dose levels 1, 5, 12.5, and 25 IU/kg, and at leastthree evaluable patients per dose level were enrolled at 50 and 100IU/kg.

After the screening (scheduled within 14 days of the FIXFc dose), thetreatment period for the patients began. The treatment period for eachdose level included a single dose of FIXFc (Day 1) up until thecompletion of the 72-hour safety observation period (3 days) for doselevels 1 and 5 IU/kg or until the last PK sample was taken for patientsin dose levels 12.5 to 100 IU/kg (approximately 10 days). Patientstreated with 1, 5, 12.5, or 25 IU/kg were enrolled and treated in asequential manner starting at 1 IU/kg. Patients receiving 50 IU/kg werenot treated on the same day and at least one day separated dosing. Aftertreatment of the 50 IU/kg patients, treatment of the 100 IU/kg patientsbegan.

The post-treatment period was a 30-day safety observation periodstarting from the day the patient received the dose of FIXFc andoverlapped with the treatment period since patients were undergoing therequired study evaluations, such as PK sampling, during this time.

Patients assigned to dose levels 12.5 to 100 IU/kg had blood samplesdrawn to assess FIX activity and FIXFc concentration. Blood samples wereto be drawn just prior to administration of FIXFc; 15 minutes followingthe end of the infusion; and at 1, 3, 6, 9, 24, 48, 72, 96, 120, 168,and 240 hours following the end of the infusion or until baseline FIXlevels were reached. If a patient continued to have FIX levels abovebaseline at the 240-hour time point (Study Day 11), samples were takenat 288 hours (Study Day 13) and again at 336 hours (Study Day 15) if theFIX level was above baseline at Study Day 13.

Patient 10 received BENEFIX™ treatment for a bleed prior to scheduledFIXFc sampling at 216 hours post dosing. Consequently, FIXFc activityand antigen data for the 216 h and following time points were excludedfrom analysis. No additional deviations occurred that are felt to haveaffected the interim analysis results of this study.

For Factor IX antigen, pharmacokinetic analyses were performed on theindividual patient observed FIXFc concentration versus time datafollowing IV infusion of FIXFc. Primary analysis was performed usingmodel-dependent methodology. FIXFc concentration data werecomputer-fitted to a two-compartment open model with elimination fromthe central compartment using user-defined initial parameter estimatesfor the calculation of initial parameter values. WinNonlin estimatedmicroscopic rate constants were generated and FIXFc concentration datawere weighted with the function of 1/(Y^(−hat)*Y^(−hat)). Observed datafor two subjects (e.g., Patients 5 and 6) were inadequately described bythe two-compartment model. Consequently, model-independent analysis wasperformed on these two patients using WinNonlin noncompartmentalanalysis IV-Infusion input model (linear trapezoidal rule for AUCcalculation). For noncompartmental analysis, the half-life wascalculated from the beta phase using the data points that describe theterminal log-linear decline in the regression. A minimum of three pointswere used to describe elimination phase. This occurred approximatelybetween 4 and 14 days. For PK analysis of antigen, the “mg/kg” doseequivalents were utilized. These values were determined based on aspecific activity for FIXFc of 60.2 IU/mg. Actual sampling times, doses,and infusion durations were used for calculations. Nominal samplingtimes and doses were used for the creation of tables andconcentration-time figures. Individual and mean PK parameters anddescriptive statistics are presented. Formal statistical analysis wasnot performed because the dose range and the number of subjects in eachcohort were too small for meaningful analysis.

For Factor IX activity, a baseline subtraction method was applied to theactivity versus time profile according the baseline subtraction decisiontree (FIG. 4). Activity values of <1% were defined at 1 IU/dL forbaseline decay. Predose times were treated as zero for the purpose ofcalculations. In addition, baseline corrected activity data weretruncated at time points that represented a return to baseline levels.Pharmacokinetic analyses were performed on the baseline subtracted FIXactivity versus time data obtained following IV infusion administrationof FIXFc. A model-dependent assessment was utilized for analysis of theIV-infusion dose groups. The baseline subtracted data werecomputer-fitted to a two-compartment open model with elimination fromthe central compartment using WinNonlin-defined parameter boundaries forthe calculation of the initial parameter values. WinNonlin estimatedmicroscopic rate constants were generated and FIXFc activity data wereweighted with the function of 1/(Y^(hat)*Y^(−hat)). Actual samplingtimes, doses, and infusion durations were used for calculations. Nominalsampling times and doses were used for the creation of tables andconcentration-time figures.

When unavailable from the actual data, the activity at 168 h post dosing(C168) and time to 1 IU/dL above baseline (TBLP1) of rFIXFc wereobtained using the WinNonlin generated microscopic rate constants tosimulate the FIXFc activity level versus time data. Individual and meanPK parameters and descriptive statistics are presented in this Example.Formal statistical analysis was not performed, because the dose rangeand the number of subjects in each cohort were too small for meaningfulanalysis.

Results for FIXFc antigen pharmacokinetics showed that FIXFc plasmaconcentrations increased sharply after the short IV infusion of FIXFc,with mean (±SD) C_(max) values of 1670 (n=1), 2730 (n=1), 7510-2480 and15400±3960 ng/mL for the 12.5, 25, 50, and 100 IU/kg nominal doselevels, respectively, and was reached within the first half-hour for allpatients All FIXFc-treated patients had dose-related increases insystemic FIXFc plasma exposure (as assessed by C_(max) and AUC_(INF)).Although limited to a single evaluable patient at the 12.5 and 25 IU/kgnominal dose, the observed increase in both C_(max) and AUC_(INF) wasreasonably proportional to dose over the dose range evaluated. (Table 3shows individual patient and group mean FIXFc antigen concentrationversus time data; sorted by nominal dose, actual dose, infusionduration, and patient number. Table 4 shows individual patient and groupmean FIXFc antigen PK summary data; sorted by nominal dose, actual dose,“mg/kg” equivalent dose, and patient number, shows individual patientand group mean FIXFc antigen PK summary data; sorted by nominal dose,actual dose, “mg/kg” equivalent dose, and patient number, and see Table11.)

FIXFc plasma concentrations declined in a biexponential fashionfollowing the short IV infusion. Both distribution (alpha) andelimination (beta) half-life values appeared to be dose-independent overthe dose range evaluated with individual patient alpha and betahalf-life values ranging from 9.79 to 21.2 hours and 71.0 to 140 hours,respectively. Mean alpha half-life values (±SD) for the 50 and 100 IU/kgnominal dose levels were 13.1±4.77 and 12.1±2.33 hours, respectively.Mean beta half-life values (±SD) for the 50 and 100 IU/kg nominal doselevels were 110±26.5 and 95.8±11.1 hours, respectively. In addition,primary PK parameter values for Cl, Vss, and MRT were determined and, ingeneral, all appeared to be dose-independent over the dose rangeevaluated. As indicated, this assessment is limited by single patientdata at the 12.5 and 25 IU/kg nominal dose levels. (Table 12 and FIGS.2, 7, and 8.)

Further, mean Cl values were 2.28±0.374 and 2.11±0.464 mL/h/kg for the50 and 100 IU/kg nominal dose levels, respectively. Mean V_(SS) valueswere 259±78.5 and 238±52.2 mL/kg for the 50 and 100 IU/kg nominal doselevels, respectively. In addition, mean MRT values were 112±21.5 and114±17.1 h for the 50 and 100 IU/kg nominal dose levels.

Results for baseline corrected FIXFc activity pharmacokinetics showedthat FIXFc activity increased sharply after the short IV infusion ofFIXFc, with mean (±SD) model-predicted C. values of 11.9 (n=1), 19.9(n=11), 41.6±8.97 and 98.2±8.21 IU/dL for the 12.5, 25, 50, and 100IU/kg nominal dose levels, respectively, and was reached within thefirst half-hour for all patients. (Table 5 shows individual patient andgroup mean baseline corrected FIXFc activity versus time data; sorted bynominal dose, actual dose, infusion duration, and patient number andTable 6 shows individual patient and group mean FIXFc activity PKsummary data; sorted by nominal dose, actual dose, “mg/kg” equivalentdose, and patient number.)

All FIXFc-treated patients had dose-related increases in FIX activity(relative to predose baseline response). Although limited to a singleevaluable patient at both the 12.5 and 25 IU/kg nominal dose levels, theobserved increase in both C_(max) and AUC_(INF) was reasonablyproportional to dose over the dose range evaluated. (Tables 6, 9, and 13and FIGS. 3 and 5.)

After the end of the infusion, the decline in baseline corrected FIXactivity exhibited biexponential decay; characterized by a rapiddistribution (alpha) phase followed by a log-linear elimination (beta)phase. During the alpha phase, the rate of decline in FIXFc activity wasvariable with individual patient alpha half-life values ranging from0.140 to 16.6 hours. The seemingly dose-dependent increase in mean alphahalf-life values was confounded by a single patient at the 12.5 and 25IU/kg nominal dose levels. In contrast, elimination (beta) half-lifevalues appeared to be dose-independent over the dose range withindividual patient beta half-life values ranging from 42.1 to 67.4 hoursover the 25 to 100 IU/kg dose range. Although estimated and reported,the elimination half-life for patient 1 treated with 12.5 IU/kg ofrFIXFc are not included in summary evaluation due to this patient's FIXlevels being detectable for only up to 96 hours resulting in a truncatedterminal phase and contributing to an underestimation of the terminalelimination half-life. Mean beta half-life values (±SD) for the 50 and100 IU/kg nominal dose levels were 52.1±10.4 and 52.5±10.1 hours,respectively, and 52.5±9.2 (range 40-67.4) hours for combined 25, 50 and100 IU/kg nominal doses. (Tables 6, 8 and 13).

In addition, primary PK parameter values for Cl, V₁, V_(SS), and MRTwere determined and, in general, all appeared to be dose-independentover the dose range evaluated.

Further, mean Cl values were 3.77±1.12 and 2.89±0.615 mL/h/kg for the 50and 100 IU/kg nominal dose levels, respectively, and 3.36±0.928 mL/h/kgfor the combined 25, 50, and 100 IU/kg nominal doses. (Tables 6, 8 and13.)

Mean V_(SS) values were 264±77.6 and 179±31.1 mL/kg for the 50 and 100IU/kg nominal dose levels, respectively, and 226±69.8 mL/kg for thecombined 25, 50, and 100 IU/kg nominal doses. (Tables 6, 8 and 13.) Inaddition, mean MRT values were 71.7±13.0 and 62.8±8.82 h for the 50 and100 IU/kg nominal dose levels, respectively, and 68.05±11.16 h for thecombined 25, 50, and 100 IU/kg nominal doses. (Tables 6, 8 and 13.)

In addition to the primary PK parameters, secondary PK parameters (e.g.,C168, K-values, IVR, etc.) were determined to evaluate FIXFc duration ofeffect. As anticipated, dose-dependent increases in C168, TBLP1, TBLP3,and TBLP5 values were observed. In contrast, K-values and IVR valuesappeared to be dose-independent over the dose range evaluated. Over thefull dose range, individual patient model-predicted and observedK-values ranged from 0.61 to 1.02 and 0.62 to 1.17 IU/dL per IU/kg,respectively. Mean model-predicted K-values for the 50 and 100 IU/kgnominal dose levels were 0.76 and 0.90 IU/dL per IU/kg, respectively,and 0.821±0.1387 (range 0.61-1.02) IU/dL per 1 IU/kg for combined 25,50, and 100 IU/kg nominal doses. Mean model-predicted IVR values for the50 and 100 IU/kg nominal dose levels were 34.5 and 35.1%, respectively.Mean observed K-values for the 50 and 100 IU/kg nominal dose levels were0.86 and 1.02 IU/dL per IU/kg, respectively, and 0.926±0.1787 (range0.97-1.17) IU/dL per 1 IU/kg for combined 25, 50, and 100 IU/kg nominaldoses. Mean observed IVR values for the 50 and 100 IU/kg nominal doselevels were 39.2 and 39.8%, respectively. (Tables 6, 7, 8 and 13.) Table7A-7B show 7 shows individual patient and group mean FIXFc activitysecondary PK summary data; sorted by nominal dose, actual dose, andpatient number.

Each 1 IU/kg of infused rFIXFc raised plasma FIX activity by 0.93±0.18IU/dl on average, and this incremental recovery (K value) showed weakpositive correlation with body weight (R²=0.336, p=0.048) (FIG. 23).

Pharmacokinetic estimates for FIXFc activity were consistent with thosefor rFIXFc antigen (e.g., compare Tables 13 and 14). Further, there wasexcellent correlation between rFIXFc activity and antigen levels,indicating the preservation of rFIXFc in vivo activity. (FIG. 9.) Inaddition, relative to historical data for BENEFIX™ (Wyeth), rFIXFcdemonstrated (Table 8) the following:

Dose linearity from 25-100 IU/kg

3 fold increase in t_(1/2beta)

3 fold increase in mean residence time

24% improved incremental recovery

2.5 fold reduced clearance

FIXFc is a recombinant fusion protein comprised of FIX attached to theFc domain of human IgG1. FIXFc has been designed to be a long-actingversion of FIX. Preclinical studies with FIXFc have shown a prolongationof the half-life of FIX activity compared to BENEFIX™, the commerciallyavailable recombinant FIX product. The rationale for this study was toevaluate the safety and PK of FIXFc in severe hemophilia B patients. Forthis study, 12 evaluable subjects aged 18 to 76 years were available forPK evaluation. Each subject received a single administration of FIXFc ata nominal dose of 12.5, 25, 50, or 100 IU/kg of body weight infusedintravenously over approximately 10 minutes. Plasma samples for PKassessments of both FIXFc activity and antigen concentrations wereobtained before infusion as well as up to 14 days after dosing. The PKof both FIXFc antigen and activity were independently characterized inthis study using model-dependent and model-independent methods.

FIXFc was well tolerated following administration of single IV doses of12.5, 25, 50, and 100 IU/kg of body weight. There was no evidence ofdrug-related serious adverse events in this study. No neutralizing orbinding antibodies to rFIXFc were detected in any subject.

Approximate dose-proportional increases in C_(max) and AUC_(INF) wereobserved for both FIXFc antigen and activity following theadministration of doses of 12.5 through 100 IU/kg, but the V and Cl weresimilar across all doses. These results indicate that FIXFc antigen andactivity exhibited linear PK over the dose range evaluated. Therelatively small V parameter values may indicate that FIXFc enters theinterstitial fluid but does not cross the cell membrane into theintracellular fluids.

Peak plasma levels of FIXFc antigen and activity were observed within0.5 h after the end of the infusion and remained detectable for severaldays after dosing. Evidence of reduced clearance and prolonged half-lifewas observed for both FIXFc antigen and activity.

Mean clearance and terminal elimination half-life values associated withFIXFc antigen concentrations for the 50 and 100 IU/kg dose levels were2.28 and 2.11 mL/h/kg and 110 and 95.8 hours, respectively. Similarly,mean clearance and terminal elimination half-life values associated withFIXFc activity levels over the same dose range were 3.77 and 2.89mL/h/kg and 52.1 and 52.5 hours, respectively. Comparison of FIXFcactivity PK results observed in the current study to reported PK forBENEFIX™ activity (Summary of Product Characteristics of BENEFIX™; Nov.18, 2009) revealed an approximate 3-fold reduction in FIXFc clearanceand an approximate 3-fold increase in both FIXFc terminal eliminationhalf-life and mean residence time relative to BENEFIX™.

With the observed improvements in PK, FIXFc will provide a prolongedprotection from bleeding, allowing less frequent injections forindividuals with Hemophilia B. Based on the results of this trial,rFIXFc may be dosed every two weeks or twice monthly using doses of 100IU/kg and at least weekly using lower doses. Such a regimen requiresfewer injections. In addition, the use of rFIXFc will have otherpotential clinical impacts such as: central venous access; improvedregimen compliance; reduced break through bleeds; and increasedprotection of joints from bleeds.

EXAMPLE 2 B-LONG Phase 1/2/3 Trial

This will be an open-label, multicenter evaluation of the safety,pharmacokinetics, and efficacy of recombinant, long-acting coagulantFactor IX Fc fusion (rFIXFc) in the prevention and treatment of bleedingin previously treated subjects with severe hemophilia B. Treatment withFIX products currently on the market necessitates dosing 2-3 times perweek. A product with a prolonged half-life that extends the requireddosing interval to once weekly or longer would be considered by themedical community as a significant improvement for the treatment ofsevere hemophilia patients.

Dose levels vary widely for rFIX products in clinical prophylaxisstudies: the reported doses range from 10 to 171 IU/kg (Roth et al.,Blood 98:3600 (2001)) or 40 to 100 IU/kg (MASAC Recommendation 177,National Hemophilia Foundation (October 2006)). Moreover, trough levelsof FIX activity during prophylaxis treatment in subjects with noclinical signs of bleeding are predicted to range between 0.2 and 3.8IU/dL (Carlsson et al., Hemophilia 4:83 (1998)). Considering theinter-individual patient variability, individualized dosage regimensbased on the clinical status of a patient are common practice.

The results of a Phase 1/2a study (Example 1) evaluating the safety andpharmacokinetics of a single dose of a frozen liquid formulation ofrFIXFc have demonstrated the drug is well tolerated at doses rangingfrom 1 to 100 IU/kg and the PK characterization suggests severaladvantages over currently available treatments, namely a half-life andMRT that are 3-fold longer than that previously reported for BENEFIX™(61 hours vs. 19 hours). The purpose of this study is to determine thePK parameter estimates of the lyophilized rFIXFc in humansprospectively, to compare these with BENEFIX™ PK parameter estimates inhumans, and to demonstrate the efficacy of lyophilized rFIXFc in theprevention and treatment of bleeding and the safety of its repeat dosingfor previously treated subjects with severe hemophilia B.

The study will entail four arms: a low dose prophylaxis regimen (n=25),a high dose prophylaxis regimen (n=25), an on-demand regimen (n=20) anda major surgery regimen (n=5). The low dose regimen arm will include aPK subgroup (n=16) dosed with BENEFIX™, followed by crossover to rFIXFc.

The primary objectives of the study are: to evaluate the safety andtolerability of rFIXFc in all treatment arms; to evaluate the efficacyof rFIXFc in all treatment arms; and to evaluate the effectiveness ofprophylaxis over on-demand therapy (comparison of the annualized numberof bleeding episodes between Arms 1 and 2 versus on-demand regimen Arm3).

The secondary objectives of the study are: to compare the PK parameterestimates of rFIXFc and BENEFIX™; to evaluate the efficacy of rFIXFc inthe on-demand and surgical arms; to evaluate and compare the PKparameter estimates of rFIXFc at baseline and Week 26 (±1 week) in thePK subgroup; to evaluate subjects' response to treatment in all arms;and to evaluate rFIXFc consumption in all arms.

Main Inclusion Criteria:

-   -   Male and 12 years of age and older and weigh at least 40 kg    -   Diagnosed with hemophilia B (baseline Factor IX level less than        or equal to 2%)    -   History of at least 100 exposure days to any Factor IX product    -   Platelet count ≧2100,000 cells/μL    -   INR (international normalized ratio)≦1.40 as defined by the        testing laboratory's normal range    -   CD4 count ≧200 cells/μL

Main Exclusion Criteria:

-   -   History of Factor IX inhibitors    -   Kidney or liver dysfunction    -   Diagnosed with another coagulation defect other than hemophilia        B    -   Prior history of anaphylaxis associated with any FIX or IV        immunoglobulin    -   administration    -   Taking systemic immunosuppressive drugs (e.g., systemic        corticosteriods;    -   however, HAART (highly active antiretroviral therapy) is        permitted)

EXAMPLE 3 FIXFc Production in HEK293 Cells

FIXFc was produced in stably transfected HEK293 cells containing anexpression cassette for FIXFc (native FIX fused directly to the Fcregion) and an expression cassette for Fc alone. The cells also weretransfected with an expression cassette for PC5, which is a processingenzyme that allows for full processing of the FIX propeptide. Thetransfected cells were grown in serum-free suspension media containingvitamin K, and they secreted three proteins: FIXFc dimer, FIXFc monomer(one FIXFc chain and one Fc chain), and Fc dimer. FIXFc monomer(“FIXFc”) was purified by column chromatography (Protein A, FractogelDEAE, and Q Sepharose pseudo-affinity elution with low ionic strengthCaCl₂), and viral inactivated and filtered for administration to humansubjects. Also see Peters et al., Blood. 2010 Mar. 11; 115(10):2057-64(Epub 2010 Jan. 7); and U.S. Pat. No. 7,566,565; each of which isincorporated by reference herein in its entirety.

Coagulant activity of FIXFc was measured by quantitating its ability torestore the clotting activity of FIX-deficient plasma using an MLAElectra 160° C. (Medical Laboratory Automation/Instrument Labs,Pleasantville, N.Y.). Results were compared to a calibration curvegenerated using serial dilutions of a World Health Organization FIXstandard.

Serine phosphorylation and tyrosine sulfation of Factor IX are thoughtto be important for in vivo recovery. It has been reported thatMONONINE™ (plasma purified Factor IX (pdFIX) marketed by CSL Berhing)has better in vivo recovery than BENEFIX™ (recombinant FIX (rFIX)marketed by Wyeth) because of the higher phosphorylation/sulfation levelof MONONINE™ (>90%/>90% versus <10%/5%). However, FIXFc produced inHEK293 cells has almost no phosphorylation/sulfation (<10%/4%, which isvery similar to BENEFIX™), and shows better IVR (1.0 IU/dl per IU/kg)than BENEFIX™ (0.7).

In addition, FIXFc produced as described above had a significantly lower(10-100 fold) level (0.01-0.001%) of activated FIX (FIXa), a productrelated impurity, than either MONONINE™ (pdFIX) or BENEFIX™ (rFIX)(0.1%). The resulting FIXFc will have fewer unwanted thrombotic eventsupon administration than MONONINE™ or BENEFIX™.

EXAMPLE 4 Pediatric Studies: Extrapolation and Interrelation Between theDevelopment in Adult and Pediatric Populations

Patient characteristics that show relationships with FIXpharmacokinetics include age-dependent physiological changes (Bjorkmanand Berntorp, Clin. Pharmacokinetics 40:815-32 (2001); and Bjorkman,Hemophilia 9(suppl 1):101-10 (2003)) and body size and composition(Shapiro, Hemophilia 11:571-82 (2005)). Thus, weight-adjusted clearance(CL) of FIX has generally been found to decrease with age and/or bodyweight during growth from infancy to adulthood, with a correspondingincrease in terminal half-life (t_(1/2)). For rFIX product (BENEFIX™),CL and volume distribution at steady state (Vss) are increased inchildren and then remain constant during adulthood; thus, theseparameters will be closely monitored in the pediatric studies.

Peak levels of FIX procoagulant activity (FIX:C) depend on the initialvolume of distribution of FIX:C after single and/or repeated doses ofFIX. The initial distribution of FIX is rapid. However, it has beenshown that in vivo recovery (mean incremental recovery) for BENEFIX™ wastypically 30% lower than that of a monoclonal antibody purified plasmaderived coagulation factor (pdFIX) (Roth et al., Blood 98:3600-3606(2001)). Furthermore, studies with pdFIX have shown that subjects 15years of age and younger have a significantly lower recovery than thosewho are older (White et al., Thromb. Haemost. 73:779-84 (1995)).Therefore, monitoring of trough and peak levels will also be performedin the pediatric studies.

Since studies have shown that children may respond differently comparedto adults, pharmacokinetic assessments at baseline with 50 IU/kg ofrFIXFc will be performed in children with abbreviated pharmacokineticsampling.

The Phase 1/2a study (SYN-FIXFc-07-001) evaluating the safety andpharmacokinetics profile of a single intravenous administration ofrFIXFc in PTPs aged 18 years and above with severe hemophilia B wasrecently completed. Preliminary results from this initial exploration inhumans demonstrates an approximately 3-fold increase in pharmacokineticparameters (mean terminal half-life, MRT, and AUC) of rFIXFc comparedwith what has been reported in the literature for BENEFIX™ (see above).Additionally, rFIXFc was well tolerated and there were no sign ofinjection site reactions as well as no development of inhibitors.Together, these safety and pharmacokinetic results support theinitiation of a Phase 1/2/3 registrational study (998HB102 Study(B-LONG), see above) evaluating the safety, pharmacokinetics, andefficacy of rFIXFc in prevention and treatment of bleeding in 104 PTPs(with at least 100 treatment EDs to previous products) 12 years andolder with severe hemophilia B (<2%). Once sufficient safety data areavailable from the registrational study, a pediatrics program will beinitiated to further investigate the safety and efficacy of rFIXFc inchildren. The demonstration of prolonged half-life of rFIX in humanswill mean that less frequent injections will be needed for theprevention and treatment of bleeding to individuals with hemophilia B.

Phase 2/3 Pediatric PTPs Study in Previously Treated Children (<12 YearsOld)

Once the data are available on 10 PTPs (≧12 years) for 26 EDs from theregistrational study (998HB 102 Study), a Pediatric Study, phase 3 willbe initiated. This Phase 2/3 pediatric study, in PTPs who had at least50 EDs to FIX products prior to enrollment, will be conducted globallyat approximately 25 clinical sites. Approximately 25 PTPs (to ensure 20evaluable subjects), age 2-11 years with severe hemophilia B (<2 IU/dL[<2%] endogenous FIX), will be screened and selected according to thepre-defined criteria. All evaluable subjects will complete thepharmacokinetic portion of the study (PK with pre-study FIX product andthen PK with rFIXFc) and will receive weekly dosing of rFIXFc for 52weeks. This study will record incremental recovery, in vivo half-life,AUC, and clearance of rFIXFc. All subjects will undergo pharmacokineticassessment at baseline with pre-study FIX and rFIXFc and the duration ofthe study for each subject will be approximately 69 weeks, includingscreening and follow-up.

Each subject will receive 50 IU/kg of rFIXFc at baseline forpharmacokinetic assessment followed by repeated weekly dosing with 50-60IU/kg of rFIXFc. With regard to patient compliance, abbreviatedpharmacokinetic sampling will be employed for pre-study product and forrFIXFc as follows: pre dose, end of injection, 30±10 minutes, 3±1 hours,24±3 (Day 1), 72±3 (Day 3), 120±3 (Day 5), and 168±3 hours (Day 7) afterthe end of injection. In order to address immunogenicity, all subjectswill be treated with rFIXFc weekly for a minimum of 50 EDs. Safetyparameters will be included for immediate safety and tolerabilityassessment, such as: (a) vital signs (pulse, blood pressure, respiratoryrate, temperature) at pre rFIXFc injection and 30 minutes postinjection; (b) hematology and coagulation parameters; (c) clinicalchemistry; (d) frequent FIX inhibitor determinations using theNijmegen-modified Bethesda assay (immediately before first exposure, ED4[Week 4], ED12, ED24, ED36, and ED50); and (e) adverse events.

Efficacy will be assessed by evaluation of number of bleeding episodes,bleeding intervals and number of treatments and consumption of FIX perannualized year and per event.

Phase 2/3 Pediatric PUPs Study In Previously Untreated Children (0-11Years Old)

Once the data from 10 previously-treated children (2-11 years) withcomplete pharmacokinetics and 50 EDs are available in the precedingstudy, a Phase 2/3 pediatric PUPs study will be initiated. This studywill be conducted globally at approximately 60 clinical sites. Up to 30PUPs (to ensure 20 evaluable subjects) for 0 and above years with severehemophilia B (<2 IU/dL [<2%] endogenous FIX) will be screened andselected according to the pre-defined criteria.

Participation in the study will vary since the initiation treatment maybegin using rFIXFc as modified prophylaxis regimen. Per patient studyparticipation is expected to be approximately four years includingscreening and follow-up. During this time most patients are expected toachieve 50 EDs to rFIXFc. In order to address immunogenicity, allsubjects will be treated with approximately 50 EDs of rFIXFc or for upto 4 years. Safety parameters will be included for immediate safety andtolerability assessment: (a) frequent FIX inhibitor determinations usingthe Nijmegen-modified Bethesda assay; and (b) adverse events.

Efficacy will be assessed by evaluation of number of bleeding episodes,bleeding intervals and number of treatments and consumption of FIX perannualized year and per event.

EXAMPLE 5 Biochemical Characterization, Activity, and PK Analysis inNon-Human Animals

The rFIXFc produced in Example 3 was characterized for itsposttranslational modification, and the following results were obtained(see Table 15 and FIG. 11). The propeptide of rFIXFc was properlyprocessed during production. rFIXFc's gamma-carboxylation pattern wassimilar to that of rFIX. Further, total Gla/molecule (11.2±0.7) ofrFIXFc was comparable to rFIX. Because gamma-carboxylation at certainresidues is essential for FIX activity, these are important results. Inaddition, Ser 158 phosphorylation and Tyr 155 sulfation of rFIXFc werecomparable to rFIX. N-linked glycans in FIX are not fully sialylated,similar to rFIX. rFIXFc O-linked glycosylation in the first EGF domainwas the same as FIX, albeit in different relative ratios. Asp 64 ofrFIXFc had a higher degree of beta-hydroxylation than rFIX or plasmaderived FIX (pdFIX). Activated FIX was present at a much lower level inthe rFIXFc preparation than in the rFIX or pdFIX preparations, as isdiscussed in detail in Example 3.

In addition, rFIXFc was administered to various animal species todetermine its activity and PK parameters. The results are shown in Table16 and FIGS. 12-16.

EXAMPLE 6 Gamma-Carboxylation

The goals of this study were to analyze and characterize γ-carboxylationof the glutamic acids (Gla) in a preclinical lot of FIXFc material andcommercially available FIX products, to characterize the Gla content ofan enriched “peak” fraction and a high salt elution “strip” fractionoriginating from a pseudo-affinity chromatography ion-exchange step, andto further separate an enriched “peak” and a high salt elution “strip”fraction by anion-exchange HPLC and further characterize the separatedspecies.

To achieve these goals, a number of complementary analytical methodswere developed. These include amino acid analysis (AAA) using basichydrolysis to determine (total) Gla content, peptide map (LC/MS) usingLys-C peptides to determine Gla distribution, analytical anion-exchangeHPLC of intact molecules to separate isoforms, and activated partialthromboplastin time (aPTF) to determine biological activity.

The two Gla (E) containing peptides are:

-   -   K1K2: YNSGKL⁷E⁸EFVQGNL¹⁵ER¹⁷ECM²⁰E²¹EK        -   [M+H]+6 Gla=2953.9        -   [M+H]+5 Gla=2909.9    -   K3: CSF²⁷EAR³⁰EVF³³ENT³⁶ERTT⁴⁰EFWK        -   [M+H]+6 Gla=2959.9        -   [M+H]+5 Gla=2915.9        -   [M+H]+4 Gla=2871.9

Thirty micrograms of sample (originating from the enriched peakfraction, high salt strip fraction and each species from the analyticalanion-exchange HPLC) was denatured, reduced, alkylated and digested withLys-C (1:20, E:S). The digest was quenched with 2% TFA and injected ontoa Jupiter C18 (2.0×250 mm) Phenomenex column. Separation was performedon an Agilent 1100 system. The column was maintained at 25° C. andpeptides were eluted with a multi-step acetonitrile gradient. Massspectrometry (Thermo-Fisher LCQ) was performed in “Triple Play” mode.

Complementary methods were developed to analyze and characterize the Glacontent and distribution of preclinical rFIXFc material. Theγ-carboxylation of glutamic acids (Gla) content and distribution in apreclinical lot of rFIXFc (enriched peak fraction) was performed andcompared to commercially available products. Analysis demonstratedsimilar Gla content and distribution with respect to commerciallyavailable products. A high salt elution “strip” fraction was analyzedand compared to the enriched peak fraction. Analysis indicated a reducedlevel of γ-carboxylation.

The FIXFc (Enriched Peak Fraction) was isolated from pseudo-affinitychromatography ion-exchange step and further separated into 3 iso-formsby analytical anion exchange HPLC. AEX column load and separated specieswere highly γ-carboxylated. (The AEX column load is the strip fractioncollected during a high salt elution step from the pseudo-affinitychromatography ion-exchange step.) AEX column load and separated specieswere biologically active. The Gla content and distribution was similarto rFIX. The peptide map indicates distribution of 4/5/6 Gla's on the K3peptide. The peptide map indicates a high population of 6 Gla's on theK1K2 peptide and a trace level of 5 Gla's.

The FIXFc (Strip Peak Fraction) was isolated from pseudo-affinitychromatography ion-exchange step and further separated into 2 iso-formsby analytical anion exchange HPLC. AEX column load and separated specieswere reduced in γ-carboxylation level. There was reduced Gla contentrelative to FIXFc enriched peak fraction. A decreased level ofbiological activity was observed. The peptide map indicates an increasedpopulation of 5 Gla's in K1K2 relative to the enriched peak fraction andmay suggest an impact on biological activity.

References (each of which is incorporated by reference herein in itsentirety): Dumont J A, et al., Monomeric Fc Fusion Molecules inTherapeutic Abs-From Bench to Clinic, Ch. 33 p 779-795; Gillis S, etal., Protein Science (1997) 6:185; White G C, et al., J. Thrombosis andHaemostasis (1997) 78:261; Hansson K, and Stenflo J, Journal Thrombosisand Haemostasis (2005) 3:2633; and Peters R T, et al., Blood (2010)115:2057.

EXAMPLE 7 Evaluation of rFIXFc Pro-Coagulant Activity in HemB MiceBleeding Models

Comparable Potency of rFIXFc and BENEFIX™ was Demonstrated in HemB MouseWhole Blood ROTEM In Vitro and in a HemB Mouse Tail Clip Bleeding ModelIn Vivo

The ability of rFIXFc to form firm and stable clots was evaluated byRotation Thromoboelastometry (ROTEM®, Pentapharm GmbH, Munich, Germany)with Calcium Chloride as activator (NATEM). Pooled whole blood collectedvia the vena cava from HemB mice was divided into seven aliquots, whichwere spiked with rFIXFc to a final concentration of 7.4%, 0.74% and0.074% of normal plasma FIX activity, or BENEFIX™ to 10%, 1%, 0.1% ofnormal. As a negative control, a blood sample was spiked with FIXformulation buffer. A total of 10 blood pools from 5 HemB mice weregenerated to complete the assessment. The NATEM reaction was initiatedby the addition of CaCl₂. Coagulation parameters, including ClottingTime (CT), Clot Formation Time (CFT) and Alpha Angle were assessed. Themean and SD of CT, CFT and alpha angle are summarized in Table 17. Thedose responses for the three parameters are plotted in FIG. 17. Allthree parameters are comparable between rFIXFc and BENEFIX™ in the doserange tested (p>0.05 by one-way ANOVA (Kruskal-Wallis) analysis).

Acute efficacy of rFIXFc was also evaluated in HemB mouse Tail Clipbleeding model. (FIG. 18.) Male HemB mice were stratified for equalpresentation of body weight and age in different treatment groups. Priorto tail clip injury, mice were anesthetized with a cocktail of 50 mg/kgKetamine and 0.5 mg/kg Dexmedetomidine and placed on a heating pad tohelp maintain the body temperature. The tails of the mice were thenimmersed in 37° C. water for 10 minutes to dilate the lateral vein.After the vein dilation, rFIXFc, BENEFIX™ or vehicle were injected viathe tail vein and 5 min later, the distal 4 mm of the tail were then cutoff using a #11 scalpel with straight edge. The shed blood was collectedinto 13 ml of warm saline for 30 minutes and the blood loss wasquantified gravimetrically. Six rFIXFc treatment groups (720, 360, 240,120, 80, 40 IU/kg, n=15) and three BENEFIX™ treatment groups (360, 120,40 IU/kg, n=15) were tested. The individual animal's blood loss valueand dose response curve of median blood loss are shown in FIG. 19(A),and the median blood loss volume of each treatment group is summarizedin Table 18. The dose response in median blood loss volume for bothrFIXFc and BENEFIX™ are comparable (p=0.9315 by unpaired t test withWelch's correction).

To determine if the three-fold extended half-life of rFIXFc relative toBENEFIX™ resulted in prolonged efficacy of rFIXFc, the present inventorsevaluated the efficacy of rFIXFc and BENEFIX™ in both ex-vivo ROTEM®assay and Tail Vein Transection bleeding model (TVT) in HemB mice. FIG.20.

For ex vivo ROTEM®, male HemB mice received 50 IU/kg of rFIXFc or 100IU/kg of BENEFIX™ by intravenous injection. Whole blood was collectedfrom the vena cava of treated animals at 5 min, 24, 72, 96, 120, 168,and 216 hour post rFIXFc dosing (n=8 mice at each time point) or at 5min, 24, 48, 72, and 96 hour post BENEFIX™ dosing (n=4 mice/time point).Blood samples were analyzed immediately by NATEM. The mean and SD forCT, CFT, and alpha angle are shown in Table 19, and the CT, CFT andalpha-angle versus time curves are shown in FIG. 21. In comparison toBENEFIX™, rFIXFc showed comparable CT, CFT, and alpha angle at 5 min,but significantly improved CT, CFT and alpha angle after 72 hrs despitea 2-fold lower dose relative to BENEFIX™.

To evaluate the prophylactic efficacy of rFIXFc and BENEFIX™, male HemBmice were stratified for equal representation of body weight and age in9 different treatment groups. rFIXFc was administered by iv injection ata dose of 4 IU/kg, 13 IU/kg, 40 IU/kg and 120 IU/kg at 72 hours prior totail vein transaction, whereas the same doses of BENEFIX™ wasadministered at 24 hour prior to the injury. Prior to tail veintransection, mice were anesthetized with a cocktail of 50 mg/kgKetamine/0.125 mg/kg Dexmedetomidine/0.1 mg/kg Buprenex. In order toallow the mice to maintain normal activity following tail veintransection, 1 mg/kg Atipamezole solution was given to reverse theeffect of Dexmedetomidine, which immediately followed by the lateraltail vein transection with a straight edged number 11 surgical blade atan area where the diameter of the tail is approximately 3 mm. Theshedding blood was washed away with warm saline to ensure clearobservation of the wound, and the mouse was then single-housed in aclean cage with white paper bedding for the next 24 hours. The re-bleedand the physical activity were observed and recorded hourly up to 12hour post injury. Moribund mice were euthanized immediately afteridentification, and a 24 hour post injury checkup was performed tocomplete the study. The Kaplan-Meier curve for Time to Euthanasia andchart of survival rates 24 hour post TVT were shown in FIG. 22. TheLog-rank test determined that all treatment groups with higher than 4IU/kg dose are significantly better than vehicle group (p<0.001).Furthermore, survival is comparable between mice that received the samedose of rFIXFc at 72 hrs prior to injury as that of BENEFIX™ at 24 hrsprior to injury (p=0.4886, 0.9268, 0.7279 and 0.5209 for 4, 13, 40 and120 IU/kg dose groups respectively). The survival rates at 24 hour postTVT were plotted and ED50 value for each molecule were extrapolated fromthe curve, the ED50 for the two treatments are similar at 17.8 IU/kg forrFIXFc and 15.4 IU/kg for rFIX. Therefore, rFIXFc provided 3-fold longerduration of protection in HemB mice relative to a comparable dose ofBENEFIX™ as measured by survival and re-bleed following tail veintransection injury. Therefore, rFIXFc provided 3-fold longer duration ofprotection in HemB mice relative to a comparable dose of BENEFIX™ asmeasured by survival and rebleed following tail vein transection injury.

In conclusion, as the data show, whereas 15.4 IU/kg of BENEFIX™ resultedin 50% of HemB mice surviving the tail vein transection at 24 hrs postdosing, 17.8 IU/kg of rFIXFc achieved 50% survival in animals that wereinjured at 72 hrs post dosing. Therefore, rFIXFc demonstrates a 3-foldlonger prophylactic efficacy in correlation with its half-life extensionrelative to BENEFIX™. The results from the bleeding models are furthercorroborated by ex vivo ROTEM® analysis of whole blood from HemB micetreated with either 100 IU/kg of BENEFIX™ or 50 IU/kg of rFIXFc. At 5min post dosing, comparable improvement in clot formation were observedin both treatment groups. However, the major ROTEM® parameters such asthe clotting time, clot formation time and alpha-angle weresignificantly improved in rFIXFc-treated mice at 72 to 216 hrs followingdosing despite a 2-fold lower dose of rFIXFc relative to BENEFIX™.

In summary, the acute potency of rFIXFc is comparable to that ofBENEFIX™ as shown in both whole blood ROTEM® in vitro and the tail clipbleeding model in HemB mice. The prolonged prophylactic efficacy ofrFIXFc was shown in ex vivo whole blood ROTEM® from treated HemB miceand was determined to be approximately 3-fold longer in comparison toBENEFIX™ in the tail vein transection bleeding model in HemB mice. Theprolonged efficacy of rFIXFc correlates well with the 3-fold longerT_(1/2) of rFIXFc relative to BENEFIX™ previously demonstrated inpharmacokinetic study in HemB mice. Therefore, rFIXFc is fully activefor on-demand treatment while achieving significantly prolongedprophylactic protection with the potential to reduce the dosingfrequency, which are under investigation in the phase 3 study.

EXAMPLE 8 Pharmacokinetic and Pharmacodynamic Analysis of rFIXFc andBENEFIX™ Following a Single Subcutaneous Dose in FIX-Deficient Mice

The pharmacokinetic (PK) and pharmacodynamic (PD) profiles ofrecombinant Factor IX-Fc (rFIXFc) and BENEFIX™ (rFIX) were determinedfollowing a single intravenous or subcutaneous injection of 200 or 400IU/kg in FIX-deficient mice. Whole blood was collected via vena cava(n=4 mice/timepoint/treatment). The concentrations of rFIXFc andBENEFIX™ in plasma were determined using a human FIX-specific ELISA. Theactivities of rFIXFc and BENEFIX™ were determined using an activatedpartial thromboplastin time (aPTT) assay. PK analyses were performedusing model-dependent methodology using WinNonLin. Results are shown inTables 22 and 23.

For FIXFc, the bioavailability in FIX-deficient mice was 38% for the 200IU/kg dose and 38-46% for the combined dose (antigen ELISA) and 29% forthe 200 IU/kg dose and 29-39% for the combined dose (aPTT activityassay) compared to rFIX, 23% and 19%, respectively. The rFIXFc had1.5-1.7 fold (200 IU/kg dose) and 1.5-2.5 fold (combined doses) improvedbioavailability compared to BENEFIX™.

For rFIXFc, the terminal half-life (antigen ELISA) was 62 hr for the 200IU/kg dose and 51-62 hr for the combined doses and the terminalhalf-life (aPTT activity assay) was 42 hr for the 200 IU/kg dose and40-42 hr for the combined doses, whereas for BENEFIX™, the terminalhalf-life was 24 hr (antigen ELISA) for the 200 IU/kg dose and 17 hr(aPTT activity assay) for the 200 IU/kg dose. This indicates a 2.5-2.6fold (200 IU/kg dose and combined dose) improvement in half-life withrFIXFc.

In addition, as Tables 22 and 23 show, rFIXFc had 4.5-5.6 fold increasein AUC/dose and a 1.9-3.7 fold increase in Cmax/dose versus BENEFIX™.

Recombinant factor IX Fc fusion (rFIXFc) protein is a long-acting formof recombinant FIX (rFIX) that will provide less frequent dosing of rFIXfor treatment of hemophilia B. From mice to non-human primates and inhemophilia B patients, rFIXFc has an approximately 3-fold longerhalf-life versus rFIX (BENEFIX™). For prophylactic treatment,intravenous delivery of rFIX remains a burdensome delivery method,especially for children and in patients with poorly accessible veins.Subcutaneous administration of rFIX presents as a more attractivedelivery route that is less invasive and with less frequent dosing. Assuch, subcutaneous delivery of rFIXFc will cause less pain anddiscomfort than intravenous delivery and result in improved compliancedue to being easier to administer and administered in less time than anintravenous route. Prophylaxis regimens will also improvequality-of-life and clinical outcomes will include decreased bleedingincidences.

The concentration of rFIXFc in mouse plasma was measured using a humanFIX-specific ELISA that measured the FIX portion of the molecule and themg/kg nominal dose was used in the analysis. A summary of the PKparameters for rFIXFc and BENEFIX™ are shown in Table 20 (antigen ELISA)and Table 21 (aPTT activity assay) for n=4/group. Both analysis byantigen and activity showed that the Cmax and AUC were significantlyimproved for rFIXFc versus BENEFIX™. Using the antigen ELISA, thebioavailability (F %) was 38% for rFIXFc versus 23% for BENEFIX™.Similarly, using the aPTT activity assay, the bioavailability was 29%for rFIXFc versus 19% for BENEFIX™. Thus, rFIXFc demonstrated anincrease in bioavailability over BENEFIX™ by 1.5 to 1.6 fold.Measurements of elimination half-life showed that rFIXFc markedlyincreased the half-life whether measured by antigen (rFIXFc 62 hr versusBENEFIX™ 24 hr) or activity (rFIXFc 42 hr versus BENEFIX™ 17 hr) assays.These data show that rFIXFc had an extended half-life compared toBENEFIX™ by 2.6 to 2.5 fold.

The rFIXFc given subcutaneously to FIX-deficient mice demonstrated a PKand PD profile with increases in Cmax and AUC for rFIXFc compared toBENEFIX™. Overall, the bioavailability for rFIXFc ranged from 29%(activity) to 38% (antigen) with a half-life of 42 hr (activity) to 62hr (antigen) compared to BENEFIX™, which had bioavailability from 19-23%and half-life from 17-24%, respectively. Thus, the half-life for rFIXFcdelivered subcutaneously in FIX-deficient mice demonstrated about a 2.2(antigen) to 3.3 (activity) fold increase over currently marketed rFIXproducts given intravenously. Overall, these data support the notionthat rFIXFc delivered subcutaneously will be of clinical benefit forprophylactic treatment in hemophilia B patients.

EXAMPLE 9 Pharmacokinetic Analysis of rFIXFc Following a SingleSubcutaneous Dose in Cynomolgus Monkeys

The pharmacokinetic (PK) profile of recombinant Factor IX-Fc (rFIXFc)was studied after a single subcutaneous dose of 50 IU/kg, 100 IU/kg or200 IU/kg in cynomolgus monkeys. The concentration of rFIXFc in plasmawas measured using a FIX-specific ELISA. Primary analysis was performedusing model-dependent methodology using WinNonLin. See Tables 22-25.

Pharmacokinetic analysis of the plasma concentration versus time data(measured by FIX-specific ELISA) demonstrated that the bioavailabilityand terminal half-life were similar among doses. The bioavailabilitiesfor rFIXFc were 40% (50 IU/kg), 34% (100 IU/kg), 36% (200 IU/kg), and36-45% (combined doses) The terminal half-lives for rFIXFc were 61 hr(50 IU/kg), 45 hr (100 IU/kg), 49 hr (200 IU/kg), and 44.58 hr (combineddoses).

The concentration of rFIXFc in monkey plasma was measured using aFIX-specific ELISA that measured the FIX portion of the molecule and themg/kg nominal dose was used in the analysis. Spike and recovery analysisdemonstrated the accuracy of this FIX-specific ELISA assay for detectingrFIXFc over the range of plasma concentrations assessed. A summary ofthe PK parameters for rFIXFc are shown in Table 22 (50 IU/kg), Table 23(100 IU/kg) and Table 24 (200 IU/kg) for n=3/group. For rFIXFc SC, thegeometric means and CV % of the geometric mean for Cmax were 860+22 (50IU/kg), 1630+97 (100 IU/kg) and 3,750+26 (200 IU/kg), respectivelyindicating a dose-dependent increase. Similar increases were seen forAUC. The geometric means for bioavailability (F %) were 40+16 (50IU/kg), 30+75 (100 IU/kg) and 36+27 (200 IU/kg), demonstrating thatbioavailability was similar among doses. Measurements of terminalhalf-life showed that the half-life was similar among doses at 58+39 hr(50 IU/kg), 45+13 hr (100 IU/kg) and 46+44 hr (200 IU/kg).

The rFIXFc given subcutaneously to cynomolgus monkeys demonstrated a PKprofile with dose-dependent increases in Cmax and AUC. Overall, thebioavailability ranged from 30-40% with a half-life of 45-58 hr. Thus,the half-life for rFIXFc delivered subcutaneously in monkeysdemonstrated about a 2.8-fold increase over currently marketed rFIXproducts given intravenously. Overall, these data support the notionthat rFIXFc delivered subcutaneously will be of clinical benefit forprophylactic treatment in hemophilia B patients.

EXAMPLE 10 Predicted Prophylactic Dosing Regimens

In comparison with the standard recommended dose regimen of 25 to 40IU/kg of FIX twice or three times weekly, the median rFIXFc activity PKresults from the Phase 1/2a study described above suggest that aboutonce weekly dosing of rFIXFc at about 22.5 IU/kg, or about every 10 daysat about 45 IU/kg, or about every 2 weeks at about 120 IU/kg issufficient to maintain a trough of 1% above baseline (FIG. 24). Thesemodel simulated estimates are validated by the available data from thePhase 1/2a trial, which fall entirely within the 95% confidence intervalof the simulated activity-over-time curve. These regimens will oftenserve at the beginning of therapy. Considering the heterogeneity ofreported clinical breakthrough bleeding events relative to trough levelof plasma FIX activity, maintenance doses will need to be adjustedindividually.

After recalculation of the PK results from the Phase 1/2 study (seeExample 11), the new predicted dosing regimen, e.g., for prophylaxis, is20 IU/kg once weekly, 40 IU/kg every 10 days, or 100 IU/kg every twoweeks (twice monthly). See also Table 27 and FIG. 25.

EXAMPLE 11 Recalculation of Pharmacokinetic Data from First in Human(FiH) Study (Example 1)

Subjects with a variety of hemophilia B genotypes, such as stopcodon/nonsense and missense mutations, were included in the FiH studydiscussed in Example 1. Several subjects had markedly reduced endogenousFIX antigen levels which correlated with markedly reduced FIX activity,while a few subjects with missense genotypes had more antigen thanmeasured activity, indicating a dysfunctional circulating protein. Thepre-treatment FIX activity in 2 subjects exceeded 2 IU/dL, likely due toan incomplete washout from their last infusion of HX concentrate basedon historical testing and disease phenotype. Based on this information,the PK data from Example 1 was recalculated without baselinesubtraction, as is described below in detail. See Table 27.

In contrast to the PK calculations (based on activity) in Example 1, ifthe rFIXFc activity PK is modeled without baseline subtraction, as wasrecently reported for the PK analysis of a glycoPEGylated rFIX (Negrieret al., Blood DOI 10.1182/blood 2011 02 335596 (2011), which is hereinincorporated by reference in its entirety), the resulting estimates ofelimination half-life and MRT are much longer than the estimates inExample 1, at 82.2±21.6 and 96.8±22.0 hours (mean±SD), respectively.However, with the knowledge that not all severe hemophilia B patientshave 0% endogenous FIX activity, and taking into account patient'sgenotype and endogenous FIX antigen level, the present inventors adopteda baseline subtraction analysis method in their PK modeling.Specifically, (a) the baseline in two patients was defined as 0% becausetheir pretreatment FIX activity was <1%, they had no detectable FIXantigen and had nonsense genotypes, (b) the baseline for three patientswas set at 0.5% because their pretreatment FIX activity was <1% and theyhad detectable FIX antigen, (c) for patients whose pretreatment FIXactivity was between 1-2%, Cmin (the lowest activity throughout the PKstudy) was defined as baseline, and (d) for patients whose pretreatmentFIX activity was ≧2%, 2% (which was the upper limit for enrollment intothe trial) was the baseline. Activity above the baseline pre-dosing wasconsidered residue drug from prior treatment, and was decayed tobaseline and subtracted from the PK data following rFIXFc dosing.

The resulting mean terminal half-life (56.7±10.9 hours, range 42.4-74.5hours) and MRT (71.8±10 hours, range 53.2-85.9 hours) of rFIXFc areapproximately 3-fold longer than that reported for rFIX. The reportedterminal half-life of rFIX is 19.3±4.97 hours (range 11.1-36.4 hours)and MRT 26.0±6.07 hours (range 15.8-46.1 hours). Roth et al., Blood98:3600-3606 (2001); and Summary of Product Characteristics forBENEFIX™, Electronic Medicines Compendium (2010)(http://www.medicines.org.uk/emc/medicine/20376/SPC/BENEFIX™/#PHARMACODYNAMIC_PROPS), each of which his incorporated herein by reference in itsentirety. Thus, the ranges for rFIXFc do not overlap the ranges forrFIX. Similarly, the mean CL of rFIXFc activity (3.18±0.78 mL/hr/kg,range 2.05-4.18 mL/hr/kg) is approximately 2.6-fold less than thatreported for rFIX (8.40±2.01 mL/hr/kg, range 4.66-13.64 mL/hr/kg), whilethe Vss of both proteins are comparable at 4-5 times the plasma volume.

Although the same trend toward improvement was observed in the rFIXFcantigen PK, both the T_(1/2α) and T_(1/2β) of rFIXFc antigen weresignificantly longer than that derived from FIX activity measurements.The T_(1/2α) estimated for rFIXFc antigen clearly deviates from thatnormally associated with FIX (2-3 hours). Furthermore, the probableincomplete washout from the pre-study replacement therapy beforeinfusion of rFIXFc sometimes resulted in a higher baseline value, whichin turn could lead to an underestimation of the rFIXFc T_(1/2)β, asmeasured by FIX activity. A number of subjects had an aPTT activity upto 3 IU/dL, well above the limit of quantification (1 IU/dL) for theaPTT assay, at later time points up to 336 hrs (14 days) post-dose.However, these time points were excluded from the estimation of theterminal half-life because the values were at or only slightly abovepretreatment baselines, thus deemed to have returned to baseline. Incontrast, the low but detectable terminal levels of rFIXFc may beunmasked by the specific and highly sensitive rFIXFc antigen ELISA,which detects as low as 0.1 IU/dL as compared to aPTT lower limit of 1.0IU/dl.

The remaining PK parameters (activity) changed a small amount relativeto elimination half-life and MRT. See Table 27(B). A dose-proportional,linear increase in FIX activity was observed based on C_(max) occurringimmediately after infusion and AUC_(INF) (Table 4). FIX activityexhibited biexponential decay following infusion of rFIXFc, and wascharacterized by a rapid distribution (alpha) phase followed by alog-linear elimination (beta) phase. The mean distribution half-life(T_(1/2)α) was highly variable for individual subjects (mean of 3.4 and10.3 hours for the two higher dose groups) (Table 27(B)). The meanelimination half-life (T_(1/2)β) was dose independent over thetherapeutic dose range tested, i.e., 53.5 hours, 57.5±8.2 hours, and56.5±14.1 hours at 25 IU/kg, 50 IU/kg, and 100 IU/kg, respectively. Thetime to 1% (1 IU/dL) above baseline, an assessment of rFIXFc activity,showed a dose-proportional increase. It was 7.3, 10.1±1.5, and 12.3±2.5days for doses of 25, 50, and 100 IU/kg, respectively. At 168 hours (1week) post dose, the plasma FIX activity was sustained at 1.1 IU/dL,2.5±0.9 IU/dL, and 4.6±1.7 IU/dL above baseline for the 25, 50, and 100IU/kg dose groups, respectively. Also dose-independent were MRT, CL, andVss over the dose range of 25 to 100 IU/kg. Furthermore, each 1 IU/kg ofinfused rFIXFc raised plasma FIX activity by 0.93±0.18 IU/dL on average(Table 27(B)), and this incremental recovery (K) showed weak positivecorrelation with body weight (R²=0.336, p=0.048)

Long-term empirical clinical experience has suggested that a sustainedplasma factor activity as low as 1 to 2 IU/dL will be adequate toprevent spontaneous bleeding events in severe hemophilia A and Bpatients, (Nilsson et al., J. Intern. Med. 232:25-32 (1992), which isherein incorporated by reference in its entirety), and increasedbleeding events are associated with the amount of time under 1% ofnormal FVIII activity. Collins et al., Thromb Haemost 7:413-420 (2009),which is herein incorporated by reference in its entirety. Thus, PKanalyses provide a means to optimize prophylactic treatment withindividualized dose modeling to achieve sustained trough levels above 1%(1 IU/dL) of baseline, reduce peak/trough variation, and improve thecost effectiveness of treatment. Carlsson et al., Haemophilia 4:83-88(1998); Kisker et al., Haemophilia 9:279-284 (2003), each of which isherein incorporated by reference in its entirety.

To construct the concentration-time profiles following different dosingregimens, Monte Carlo simulation was conducted using the population PKmodel of rFIXFc. The mean estimates of model parameters (CL, volume ofdistribution, inter-compartmental clearance, and volume of the secondcompartment) in the tested population, the inter-individual variance,and the residual variability were adopted for this Phase1/2a study. Wanget al., J. Clin. Pharmacol. 49:1012-1024 (2009), which is hereinincorporated by reference in its entirely. One thousand subjects weresimulated per dosing regimen with 14 to 16 sampling points for eachsubject. There were 14 sampling points for weekly dosing, 15 for every10 day dosing, and 16 for every other week dosing. The body weight (BW)was generated according to the published method, Wang et al. (2009).i.e., based on a power equation of Z=BW−0.5. The median BW in 1000subjects was assumed to be 75 kg. Based on the simulatedconcentration-time profiles, the mean±standard deviation (SD) of thedrug concentration-time profiles of the 1000 subjects was constructedgraphically for different dosing regimens. FIG. 25.

In comparison with the standard recommended dose regimen of 25 to 40IU/kg of FIX twice weekly, the median rFIXFc activity PK modelingresults from this study show that once weekly dosing of rFIXFc at 20IU/kg, or every 10 days at 40 IU/kg, or every 2 weeks at 100 IU/kg issufficient to maintain a trough of 1% above baseline. FIG. 25. Thesemodel-simulated estimates are validated by the available data from thisPhase 1/2a study, which fall entirely within the 95% confidence intervalof the simulated activity-over-time curve. However, considering theheterogeneity of reported clinical breakthrough bleeding events relativeto trough level of plasma FIX activity (Bjorkman, Haemophilia 9:101-110(2003); Ahnstrom et al., Haemophilia 10:689-697 (2004), each of which isherein incorporated by reference in its entirety), the maintenance dosewould likely require individual adjustment.

TABLES

TABLE 1 Polynucleotide Sequences: FIX-FcA. FIX-Fc Chain DNA Sequence (SEQ ID NO: 1, which encodes SEQ ID NO: 2)pSYN-FIX-030 Nucleotide sequence (nt 1 to 7583):FIX exon 1 (signal peptide, 1st amino acid propeptide): nt 690-777FIX mini intron: nt 778-1076 FIX propeptide sequence: nt 1077-1126Mature FIX sequence: nt 1127-2371 Fc: nt 2372-3052gcgcgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttcgcgacgtacggccgccaccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgtacaggtttgtttccttttttaaaatacattgagtatgcttgccttttagatatagaaatatctgatgctgtcttcttcactaaattttgattacatgatttgacagcaatattgaagagtctaacagccagcacgcaggttggtaagtactgtgggaacatcacagattttggctccatgccctaaagagaaattggctttCagattatttggattaaaaacaaagactttcttaagagatgtaaaattttcatgatgttttcttttttgctaaaactaaagaattattcttttacatttcagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaatctagagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccgtgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagcacccaatcatttaatgacttcactcgggttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacacctatttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtaccttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtgtcccggtatgtcaactggattaaggaaaaaacaaagctcactgacaaaactcacacatgcccaccgtgcccagctccggaactcctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagaattcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttggggtgggcgaagaactccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccgaagcccaacctttcatagaaggcggcggtggaatcgaaatctcgtagcacgtgtcagtcctgctcctcggccacgaagtgcacgcagttgccggccgggtcgcgcagggcgaactcccgcccccacggctgctcgccgatctcggtcatggccggcccggaggcgtcccggaagttcgtggacacgacctccgaccactcggcgtacagctcgtccaggccgcgcacccacacccaggccagggtgttgtccggcaccacctggtcctggaccgcgctgatgaacagggtcacgtcgtcccggaccacaccggcgaagtcgtcctccacgaagtcccgggagaacccgagccggtcggtccagaactcgaccgctccggcgacgtcgcgcgcggtgagcaccggaacggcactggtcaacttggccatggtttagttcctcaccttgtcgtattatactatgccgatatactatgccgatgattaattgtcaacacgtgctgatcagatccgaaaatggatatacaagctcccgggagctttttgcaaaagcctaggcctccaaaaaagcctcctcactacttctggaatagctcagaggcagaggcggcctcggcctctgcataaataaaaaaaattagtcagccatggggcggagaatgggcggaactgggcggagttaggggcgggatgggcggagttaggggcgggactatggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacaccctcgtcgagctagcttcgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctccagtacgtgattcttgatcccgagctggagccaggggcgggccttgcgctttaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaggatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctccagggggctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaggggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctggagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaacacgtggtcgcggccgcgccgccaccatggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgcgatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgactcgagagatctggccggctgggcccgtttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggtcatcatcaccatcaccattgagtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagtggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctagaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccaB. Fc DNA sequence (mouse IgK signal peptide underlined)(SEQ ID NO: 3, whichencodes SEQ ID NO: 4) This is the Fc cassette from pSYN-FIX-030. In addition,there is a separate Fc expression cassette that was transfected into the cellline in plasmid pSYN-Fc-015 that encodes the same amino acid sequence butcontains a few noncoding changes. The second copy of Fc encoding sequenceenables a better monomer: dimer ratio.atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgcgatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa

TABLE 2 Polypeptide SequencesFIX-Fc Monomer Hybrid: created by coexpressing FIX-Fc and Fc chains.A. FIX-Fc chain (SEQ ID NO: 2):(28 amino acid signal sequence underlined, 18 amino acidpropeptide double underlined, Fc portion in italics.) TheC-terminal lysine is not present in either subunit; thisprocessing is often observed in recombinant proteinsproduced in mammalian cell culture, as well as withplasma derived proteins. FIXFC-SC SUBUNIT: FIX Signal Peptide: −46 MQRVNMIMAE SPGLITICLL GYLLSAEC FIX Propeptide: −18  TVFLDHENAN KILNRPKR1 YNSGKLEEFV QGNLERECME EKCSFEEARE VFENTERTTE FWKQYVDGDQ 51CESNPCLNGG SCKDDINSYE CWCPFGFEGK NCELDVTCNI KNGRCEQFCK 101NSADNKVVCS CTEGYRLAEN QKSCEPAVPF PCGRVSVSQT SKLTRAETVF 151PDVDYVNSTE AETILDNITQ STQSFNDFTR VVGGEDAKPG QFPWQVVLNG 201KVDAFCGGSI VNEKWIVTAA HCVETGVKIT VVAGEHNIEE TEHTEQKRNV 251IRIIPHHNYN AAINKYNHDI ALLELDEPLV LNSYVTPICI ADKEYTNIFL 301KFGSGYVSGW GRVFHKGRSA LVLQYLRVPL VDRATCLRST KFTIYNNMFC 351AGFHEGGRDS CQGDSGGPHV TEVEGTSFLT GIISWGEECA MKGKYGIYTK 401VSRYVNWIKE KTKLT

451 TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV 501

551

601

B. Fc chain (SEQ ID NO: 4) 20 amino acid heterologous mouse Igκlight chain signal peptide (underlined): −20 METDTLLLWV LLLWVPGSTGMature Fc sequence (corresponding to human IgG1 amino acids221 to 447, EU numbering) 1DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED 51PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK 101CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK 151GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG 201NVFSCSVMHE ALHNHYTQKS LSLSPGK

TABLE 3 Individual Patient FIXFc Antigen Concentration versus Time Data;Sorted by Nominal Dose, Actual Dose, Infusion Duration, and PatientNumber Actual Time Concentration Actual Time Concentration Actual TimeConcentration Actual Time Concentration (h) (ng/mL) (h) (ng/mL) (h)(ng/mL) (h) (ng/mL) Patient 1 Patient 2 Patient 3 Patient 4 −0.50 0.0−1.23 0.0 −0.18 0.0 −0.18 0.0 0.17 2325.3 0.17 3352.1 0.28 5915.3 0.178166.5 0.42 1632.4 0.40 3017.3 0.42 6574.3 0.42 7362.3 1.17 1497.7 1.152280.7 1.17 5764.7 1.17 6723.4 3.18 1466.4 3.15 2077.5 3.17 4204.8 3.175291.4 6.13 1268.2 6.15 2054.7 6.17 3956.2 6.18 4673.1 9.12 1100.7 9.151700.4 9.17 3567.7 9.17 3954.6 24.12 805.0 24.23 1417.3 24.17 2805.624.17 3327.6 48.03 544.5 48.40 766.0 48.98 1727.7 48.20 2148.7 72.23377.7 70.73 719.0 72.40 1165.8 72.17 1632.2 96.75 215.3 92.57 480.296.98 917.1 96.17 1234.4 120.13 192.6 119.98 326.3 121.23 673.9 120.13894.0 141.95 128.6 141.10 241.1 168.65 568.2 144.18 645.2 169.45 112.4167.98 194.6 240.15 265.4 168.22 564.1 192.37 93.6 192.85 160.1 290.97286.4 192.20 509.2 216.28 76.1 216.98 149.0 337.98 238.5 216.23 474.5237.30 76.4 238.65 125.7 240.23 446.1 Patient 5 Patient 6 Patient 7Patient 8 −0.18 0.0 −0.07 0.0 −1.27 0.0 −1.37 0.0 0.17 7520.2 0.1711671.7 0.22 7055.9 0.25 27413.4 0.43 7233.9 0.42 8654.5 0.42 6215.70.47 23640.8 1.20 6752.1 1.17 8880.4 1.17 5498.6 1.35 18505.6 3.155873.1 3.17 8509.3 3.17 4477.7 3.22 15708.1 6.23 5919.2 6.17 7618.7 6.174084.8 6.17 14915.6 9.20 5332.9 9.17 6584.2 9.17 3888.9 9.17 16486.424.17 4215.9 48.17 3217.7 24.17 2849.4 24.72 9937.8 48.15 2986.6 72.171651.6 48.82 1630.6 48.90 6383.5 72.15 1933.3 96.17 1580.1 72.57 1295.772.38 4190.6 96.03 1249.0 120.17 722.7 96.57 1150.7 96.40 3774.7 120.13401.4 240.17 329.5 121.15 954.9 120.30 2514.9 144.03 482.3 288.17 292.7144.10 780.6 168.77 1626.0 168.17 478.0 336.17 252.7 168.82 447.6 240.27924.7 192.12 433.7 192.77 446.5 288.83 682.4 216.15 368.9 240.57 427.8337.03 586.4 240.07 264.0 Patient 9 Patient 10 Patient 11 Patient 12−0.82 0.0 −0.48 0.0 −0.15 0.0 −1.12 0.0 0.28 15027.1 0.25 16760.0 0.2319641.7 0.17 15194.5 0.63 13374.1 0.50 11529.0 0.47 17267.2 0.42 12255.71.17 12395.6 1.22 10566.3 1.22 15902.2 1.17 11171.3 3.20 10808.4 3.229889.0 3.22 13708.9 3.17 9835.4 6.22 9640.2 6.22 8290.2 6.25 12469.46.17 8513.2 9.15 10505.5 9.22 7114.7 9.22 12029.8 9.17 8413.0 23.156487.3 24.22 5877.0 24.22 8083.3 24.17 5538.2 46.62 5324.8 48.22 3980.447.72 4431.0 48.20 3885.5 70.10 2895.5 72.22 2455.6 71.88 2162.6 72.132959.9 94.15 3208.3 96.12 2052.6 191.72 1468.7 95.17 2215.4 118.132610.6 120.22 1302.5 263.72 428.6 119.17 1799.7 166.10 2007.2 144.221349.3 167.38 1339.7 238.15 1086.2 168.22 1221.0 239.50 892.4 286.15942.8 192.18 910.2 287.25 646.9 335.57 621.3 216.22 136.2

TABLE 4 Individual Patient and Group Mean FIXFc Antigen PharmacokineticSummary Data Nominal Actual Equivalent Alpha Beta Dose Dose Dose C_(max)AUC_(INF) CI* V_(SS)* MRT* HL* HL* (IU/kg IU/kg mg/kg Patient (ng/mL)(h*ng/mL) (mL/h/kg) (mL/kg) (h) (h) (h) 12.5 13.714 0.228 1 1670  913002.50 245  98.2 21.2 107 N 1    1 1   1 1 1 1 25 27.250 0.453 2 2730 144000 3.14 273  87.1 11.3 71.0 N 1    1 1   1 1 1 1 50 54.5 0.905 35470  356000 2.54 366  144 18.6 138 54.5 0.905 4 6910  389000 2.32 244 105 10.6 85.3 54.5 0.905 5 7520  416000 2.17 184  84.5 NC 94.3 54.5130.906 6 11700  531000 1.71 190  112 NC 140 55.878 0.928 7 5950  3480002.67 310  116 10.1 93.9 N 5    5 5   5 5 3 5 Mean 7510  408000 2.28 259 112 13.1 110 SD 2480  73900  0.374  78.5 21.5 4.77 26.5 SE 1110  33100 0.167  35.1 9.60 2.75 11.8 Geometric Mean 7230  403000 2.26 250  11112.6 108 CV % Geometric Mean 30.3     17.1 17.6   30.8 19.4 34.9 23.8100 109 1.81 10 12500  667000 2.72 263  96.8 9.79 78.0 109 1.81 8 216001200000 1.51 156  103 15.7 94.3 109 1.81 9 13400  998000 1.81 248  13711.5 107 109.176 1.81 11 17200  844000 2.15 226  105 13.0 97.1 109.4411.82 12 12500  778000 2.34 295  126 10.6 102 N 5    5 5   5 5 5 5 Mean15400  897000 2.11 238  114 12.1 95.8 SD 3960   206000^(a)   0.464^(b)  52.2^(c) 17.1 2.33 11.1 SE 1770  92000  0.208  23.3 7.64 1.04 4.96Geometric Mean 15100  878000 2.06 232  113 11.9 95.2 CV % Geometric Mean24.5     22.9 22.9   24.7 14.8 118.7 12.2 *CL, Vss, MRT, T1/2 α andT1/2β for combined 12.5-100 IU/kg doses are 2.30 ± 0.46 (1.51-2.72); 250± 58.2 (156-366); 110 ± 18.5 (84.5-144); 12.0 ± 4.0 (10.1-18.6, notincluding two patients whose PK parameters were determined bynon-compartmental analysis); and 101 ± 20.9 (78-140), respectively. Dueto correction of rounding or other errors, ^(a)should be 207,000, and^(b)should be 0.468, ^(c)should be 52.1.

TABLE 5 Individual Patient and Group Mean FIXFc Activity and BaselineCorrected FIXFc Activity versusTime Data; Sorted by Nominal Dose, ActualDose, Infusion Duration, and Patient Number Actual Baseline Time ResultCorrected Result (h) (IU/dL) (IU/dL) Patient 1 −309.80 2 NC −0.50 3 0.00.17 16 13.0 0.42 11 8.1 1.17 10 7.1 3.18 12 9.4 6.13 9 6.6 9.12 10 7.924.12 7 5.0 48.03 6 4.0 72.23 4 2.0 96.75 3 1.0 120.13 3 1.0 141.95 31.0 169.45 2 0.0 192.37 3 1.0 216.28 3 1.0 237.30 3 1.0 746.22 3 1.0Patient 2 −310.60 3 NC −1.23 2 0.0 0.17 23 21.0 0.40 19 17.0 1.15 1513.0 3.15 13 11.0 6.15 11 9.0 9.15 13 11.0 24.23 8 6.0 48.40 6 4.0 70.736 4.0 92.57 4 2.0 119.98 4 2.0 141.10 4 2.0 167.98 3 1.0 192.85 2 0.0216.98 3 1.0 238.65 3 1.0 891.90 2 0.0 Patient 3 −524.08 <1.0 NC −0.18 20.0 0.28 44 42.0 0.42 31 29.0 1.17 27 25.1 3.17 22 20.2 6.17 18 16.49.17 17 15.6 24.17 12 11.0 48.98 7 6.0 72.40 6 5.0 96.98 6 5.0 121.23 54.0 168.65 3 2.0 240.15 1 0.0 290.97 1 0.0 337.98 1 0.0 675.22 2 1.0Patient 4 −285.52 1 NC −0.18 <1.0 0.0 0.17 59 58.0 0.42 45 44.0 1.17 4039.0 3.17 30 29.0 6.18 26 25.0 9.17 22 21.0 24.17 14 13.0 48.20 9 8.072.17 8 7.0 96.17 5 4.0 120.13 4 3.0 144.18 4 3.0 168.22 3 2.0 192.20 32.0 216.23 2 1.0 240.23 2 1.0 720.73 <1.0 0.0 Patient 5 −104.18 <1.0 NC−0.18 <1.0 0.0 0.17 35 34.0 0.43 30 29.0 1.20 25 24.0 3.15 21 20.0 6.2319 18.0 9.20 NR NR 24.17 13 12.0 48.15 9 8.0 72.15 7 6.0 96.03 5 4.0120.13 4 3.0 144.03 3 2.0 168.17 2 1.0 192.12 2 1.0 216.15 2 1.0 240.072 1.0 547.07 <1.0 0.0 Patient 6 −503.20 3 NC −0.07 3 0.0 0.17 3 0.0 0.4264 61.0 1.17 57 54.1 3.17 54 51.3 6.17 42 39.6 9.17 43 40.9 24.17 2624.0 48.17 17 15.0 72.17 13 11.0 96.17 10 8.0 120.17 9 7.0 168.17 6 4.0240.17 4 2.0 288.17 3 1.0 336.17 4 2.0 504.17 3 1.0 Patient 7 −438.43<1.0 NC −1.27 4 0.0 0.22 46 42.0 0.42 38 34.1 1.17 30 26.2 3.17 28 24.56.17 24 20.8 9.17 22 19.2 24.17 14 12.4 48.82 10 9.0 72.57 6 5.0 96.57 54.0 121.15 4 3.0 114.10 3 2.0 168.82 2 1.0 192.77 2 1.0 240.57 2 1.0744.57 3 2.0 Patient 8 −120.42 <1.0 NC −1.37 <1.0 0.0 0.25 129 128.00.47 117 116.0 1.35 102 101.0 3.22 98 97.0 6.17 80 79.0 9.17 72 71.024.72 53 52.0 48.90 30 29.0 72.38 19 18.0 96.40 14 13.0 120.30 9 8.0168.77 6 5.0 240.27 3 2.0 288.83 2 1.0 337.03 2 1.0 840.28 <1.0 0.0Patient 9 −193.05 8 NC −0.82 3 0.0 0.28 100 97.0 0.63 93 90.1 1.17 9491.1 3.20 80 77.3 6.22 69 66.6 9.15 64 61.9 23.15 47 45.0 46.62 25 23.070.10 17 15.0 94.15 13 11.0 118.13 9 7.0 166.10 5 3.0 238.15 3 1.0286.15 2 0.0 335.57 2 0.0 741.77 3 1.0 Patient 10 −334.63 1 NC −0.48 20.0 0.25 120 118.0 0.50 104 102.0 1.22 84 82.1 3.22 75 73.2 6.22 60 58.49.22 56 54.6 24.22 36 35.0 48.22 21 20.0 72.22 14 13.0 96.12 11 10.0120.22 7 6.0 144.22 6 5.0 168.22 6 5.0 192.18 4 3.0 216.22 85 84.0744.95 2 1.0 Patient 11 −912.28 2 NC −0.15 2 0.0 0.23 110 108.0 0.47 106104.0 1.22 96 94.0 3.22 92 90.0 6.25 81 79.0 9.22 70 68.0 24.22 53 51.047.72 33 31.0 71.88 25 23.0 167.72 8 6.0 191.72 8 6.0 263.72 4 2.0359.72 3 1.0 383.97 3 1.0 890.97 14 12.0 Patient 12 −342.58 2 NC −1.12 20.0 0.17 108 106.0 0.42 90 88.0 1.17 70 68.0 3.17 69 67.0 6.17 55 53.09.17 55 53.0 24.17 37 35.0 48.20 25 23.0 72.13 14 12.0 95.17 10 8.0119.17 7 5.0 167.38 6 4.0 239.50 3 1.0 287.25 2 0.0 526.42 4 2.0 Note:Data in bold represent a return to baseline and were excluded fromanalysis.

TABLE 6 Individual Patient and Group Mean FIXFc Activity PharmacokineticSummary Data; Sorted by Nominal Dose, Actual. Dose, and Patient NumberAUC/ Nom- Dose inal Actual C_(max) AUC_(INF) (IU*h/ CI V₁ V_(SS) AlphaBeta Dose Dose (IU/ (h*IU/ AUC_(α) AUC_(β) dL per (mL/ (mL/ (mL/ MRT HLHL (IU/kg) (IU/kg) Patient dL) dL) (%) (%) IU/kg) h/kg) kg) kg) (h) (h)(h) 12.5 13.714 1 11.9  418 0.231 99.8 30.5 3.28 102 157 48.0 0.140 33.3N 1    1 1 1 1 1 1 1 1 1 1  25 27.25 2 19.9  753 2.50 97.8 27.6 3.62 134275 76.0 1.20 54.0 N 1    1 1 1 1 1 1 1 1 1 1 50 54.5 3 34.5 1280 5.794.5 23.5 4.26 155 365 85.8 2.32 62.9 54.5 4 48.5 1450 12.4 87.7 26.63.76 111 282 75.1 3.64 58.9 54.5 5 33.0 1190 1.5 98.3 21.8 4.58 160 27459.9 0.840 42.1 54.513 6 53.5 2960 1.0 99.1 54.3 1.84 100 149 81.1 1.0756.7 55.878 7 38.6 1270 2.2 97.9 22.7 4.41 141 248 56.4 1.07 40.0 N 5   5 5 5 5 5 5 5 5 5 5  Mean 41.6 1630 4.56 95.5 29.8 3.77 133 264 71.71.79 52.1 SD   8.97^(a)  750 4.75 4.70 13.8 1.12 26.7 77.6 13.0 1.1910.4 SE  4.01  335 2.13 2.10 6.18 0.501 11.9 34.7 5.79 0.531  4.65Geometric 40.9 1530 2.98 95.4 27.9 3.59 131 254 70.7 1.52 51.3 Mean CV %21.4    39.1 136.5 5.0 39.4 39.4 21.1 33.8 18.8 68.6 21.0 Geometric Mean100 109 10 98.9 3330 18.5 81.3 30.6 3.28 109 216 65.9 6.53 54.6 109 8111   4580 28.9 71.1 42.0 2.38 98.0 145 61.1 13.2 54.2 109 9 92.1 354017.0 82.9 32.5 3.08 118 163 53.1 9.43  42.4^(d) 109.176 11 99.1 515028.6 71.3 47.2 2.12 110 162 76.2 16.6 67.4 109.441 12 89.9 3060 9.2 90.828.0 3.58 121 207 57.9 4.19 43.8 N 5    5 5 5 5 5 5 5 5 5 5  Mean 98.23930 20.4 79.5 36.1 2.89 111 179 62.8 9.99 52.5 SD   8.21^(b)   893^(c)8.37 8.37 8.17 0.615 8.98 31.1 8.82 4.99 10.1 SE  3.67  399 3.74 3.743.65 0.275 4.02 13.9 3.95 2.23  4.51 Geometric 97.9 3860 18.9 79.1 35.42.83 111 177 62.4 8.92 51.7 Mean CV %  8.2   22.4 49.7 10.5 22.4 22.58.2 17.4 13.8 59.4 19.0 Geometric Mean Due to correction of rounding orother errors, ^(a)should be 8.98, ^(b)should be 8.23, ^(c)should be 892and ^(d)should be 42.2.

Table 7A-7B. Individual Patient and Group Mean FIXFc Activity SecondaryPharmacokinetic Summary Data; Sorted by Nominal Dose, Actual. Dose, andPatient Number Nom- In Vivo In Vivo inal Actual C168^(a) K Value^(e) KValue^(f) Recov- Recov- Dose Dose (IU/ TBLP1^(b) TBLP3^(c) TBLP5^(d)(IU/dL per (IU/dL per ery^(g) ery^(h) (IU/kg) (IU/kg) Patient dL) (Day)(Day) (Day) IU/kg) IU/kg) (%) (%) 12.5 13.714 1 0.264 4.34 2.13 1.110.87 0.95 30.8 33.6 N 1 1 1 1 1 1 1 1 25 27.25 2 1.09 7.28 3.72 2.060.73 0.77 31.8 33.5 N 1 1 1 1 1 1 1 1 50 54.5 3 2.09 9.79 5.64 3.70 0.630.77 33.0 40.2 54.5 4 2.08 9.58 5.69 3.89 0.89 1.06 37.8 45.2 54.5 51.22 7.50 4.72 3.42 0.61 0.62 33.6 34.6 54.513 6 4.61 12.2 8.47 6.720.98 1.12 38.2 43.6 55.878 7 1.17 7.37 4.74 3.51 0.69 0.75 29.9 32.6 N 55 5 5 5 5 5 5 Mean 2.23 9.29 5.85 4.25 0.76 0.86 34.5 39.2 SD 1.4 1.981.54 1.39 0.17 0.22 3.5 5.5 SE 0.627 0.886 0.687 0.623 0.074 0.0963 1.62.5 Geometric 1.96 9.12 5.71 4.10 0.75 0.84 34.4 38.9 Mean CV % 60.021.2 24.1 28.6 21.5 25.4 10.2 14.4 Geometric Mean Table 7B Nom- In VivoIn Vivo inal Actual C168^(a) K Value^(e) K Value^(f) Recov- Recov- DoseDose (IU/ TBLP1^(b) TBLP3^(c) TBLP5^(d) (IU/dL per (IU/dL per ery^(g)ery^(h) (IU/kg) (IU/kg) Patient dL) (Day) (Day) (Day) IU/kg) IU/kg) (%)(%) 100 109 10 4.08 11.6 8.01 6.34 0.91 1.08 43.4 51.8 109 8 4.88 12.18.57 6.92 1.02 1.17 28.7 33.1 109 9 3.09 9.87 7.07 5.78 0.84 0.89 39.741.8 109.176 11 6.77 14.7 10.3 8.21 0.91 0.99 27.8 30.3 109.441 12 3.099.96 7.07 5.72 0.82 0.97 35.8 42.2 N 5 5 5 5 5 5 5 5 Mean 4.38 11.6 8.206.59 0.90 1.02 35.1 39.8 SD 1.53 1.97 1.34 1.03 0.0784 0.11 6.8 8.5 SE0.685 0.881 0.597 0.459 0.0351 0.0482 3.0 3.8 Geometric 4.19 11.5 8.126.53 0.90 1.02 34.5 39.1 Mean CV % 34.1 16.5 15.7 15.0 8.6 10.5 19.821.6 Geometric Mean ^(a)C168 = Estimated FIX activity above baseline atapproximately 168 h after dose. Value in italics was estimated fromsimulations performed using a one-compartment model and patientmicroscopic rate constants. ^(b)TBLP1 = Model-predicted time after dosewhen FIX activity has declined to approximately 1 IU/dL above baseline.Values in italics were estimated from simulations performed using aone-compartment model and patient microscopic rate constants. ^(c)TBLP3= Model-predicted time after dose when FIX activity has declined toapproximately 3 IU/dL above baseline. ^(d)TBLP5 = Model-predicted timeafter dose when FIX activity has declined to approximately 5 IU/dL abovebaseline. ^(e)K-Value was calculated using model predicted C_(max) valuegenerated from background subtracted results divided by dose.^(f)K-Value was calculated using the observed maximum post dose sampleresult; K-value = (Baseline Subtracted C_(max) observed)/Dose).^(g)In-vivo Recovery = 100 × (Model Predicted C_(max) from baselinesubtracted data/Dose) × Plasma Volume (dL)/Dose in IU; where plasmavolume in mL = (23.7 × Ht in cm) + (9.0 × Wt in kg) − 1709. ^(h)In-vivoRecovery = 100 × (Baseline Subtracted Observed C_(max))× Plasma Volume(dL)/Dose in IU; where plasma volume in mL = (23.7 × Ht in cm) + (9.0 ×Wt in kg) − 1709.

TABLE 8 Phase 1/2a Study: Comparison of PK Parameters for rFIXFc andBENEFIX ™ *rFIXFc ^(†)BENEFIX ™ [Mean ± SD (min-max)] [Mean ± SD(min-max)] Parameters [N = 11] [N = 11] t_(1/2) (hours) 52.5 ± 9.2(40-67.4)  19.3 ± 4.97 (11.1-36.4)  MRT (hours) 68.05 ± 11.16(53.1-85.8)  26.0 ± 6.07 (15.81-46.09) CL (mL/ 3.36 ± 0.93 (1.84-4.58)8.4 ± 2.01 (4.66-13.64) hour/kg) Incremental  0.93 ± 0.18(0.62-1.17)^(a) 0.75 ± 0.23 (0.34-1.38)  Recovery (IU/dL per IU/kg)C_(max) (IU/dL 24 hrs post-injection per IU/kg) AUC 48 hrspost-injection *Estimates from 2-compartmental analysis of FIX activityat the nominal doses 25, 50 and 100 IU/kg (n = 11) ^(†)Summary ofProduct Characteristics of BENEFIX ™ (Nov. 18, 2009); Median and range(n = 56) ^(a)Range corrected due to rounding or other errors as0.63-1.18. Relative to Historical Data for BENEFIX ™, rFIX-Fcdemonstrated: 3x increase in half-life and mean residence time 24%improved incremental recovery relative 2.5x reduced clearance

TABLE 9 Phase 1/2a Study: Dose Proportional Increase in Cmax and AUC ofrFIXFc (activity) Dose # of Cmax (IU/dL) AUC (h*IU/dL) (IU/kg) Patients[Mean ± SD (min-max)] [Mean ± SD (min-max)] 25 1 19.9 753 50 5 41.6 ±8.97 (33.0-53.5)  1630 ± 750 (1190-2960) 100 5 98.2 ± 8.21 (89.9-111.0)3930 ± 893 (3060-5150) Also see FIG. 5.

TABLE 10A-10B Estimated Therapeutic Duration of rFIXFc at 50 and 100IU/kg Doses. Parameter Geo Median FIX:C on Day 7 2.0 IU/dL (abovebaseline) Time to 1 IU/dL above baseline 9.1 days Time to 3 IU/dL abovebaseline 5.7 days Parameter Geo Median FIX:C on Day 7 4.2 IU/dL (abovebaseline) Time to 1 IU/dL above baseline 11.5 days  Time to 3 IU/dLabove baseline 8.1 days Also see FIG. 6A-6B.

TABLE 11 Dose Proportional Increase in Cmax and AUC for rFIXFc Antigen.Dose # of Cmax (ng/mL) AUC (h*ng/mL) (IU/kg) patients [Mean ± SD] [Mean± SD] 25 1 2,730 144,000 50 5 7,510 ± 2,480 408,000 ± 73,900  100 515,400 ± 3,960  897,000 ± 206,000 Also see FIG. 7.

TABLE 12 Pharmacokinetic Estimates for rFIXFc Antigen 50 IU/kg [Mean ±SD] 100 IU/kg [Mean ± SD] Parameters (N = 5) (N = 5) CL (mL/hour/kg)2.28 ± 0.37  2.11 ± 0.46 Vss (mL/kg) 259 ± 78.5  238 ± 52.2 MRT (hours)112 ± 21.5  114 ± 17.1 t_(1/2) (hours) 110 ± 26.5 95.8 ± 11.1 Also seeFIG. 8A-8B.

TABLE 13 Mean PK Values Based on Activity AUC/Dose Cmax AUCINF AUCa AUCb(IU*h/dL C1 V1 VSS MRT t1/2α (IU/dL) (h*IU/dL) (%) (%) per IU/kg)(mL/kg) (mL/h/kg) (mL/kg) (h) (h) n 11 11 11 11 11 11 11 11 11 11 Mean65.364 2596.636 11.591 88.427 32.436 3.555 123.364 226.000 68.045 5.463Std Dev 32.9708 1497.1234 10.4490 10.5210 10.7506 0.9257 21.2804 69.758211.1637 5.4197 % CV 50.4420 57.6563 90.1479 11.8980 33.1435 27.589017.2501 30.8664 16.4063 99.2128 Median 53.500 2960.000 9.200 90.80028.000 3.580 118.000 216.000 65.900 3.640 Minimum 19.90 753.00 1.0071.10 21.80 1.84 98.00 145.00 53.10 0.84 Maximum 111.00 5150.00 28.9099.10 54.30 4.58 160.00 365.00 85.80 16.60 Geo. mean 56.951 2181.2946.781 87.826 31.020 3.226 121.767 216.533 67.218 3.326 IncrementalIncremental Recovery [5] Recovery [6] In Vivo In Vivo t1/2β C168 [1]TBLP1 [2] TBLP3 [3] TBLP5 [4] (IU/dL per IU/dL per Recovery [7] Recovery[8] (h) (IU/dL) (Day) (Day) (Day) IU/kg) IU/kg) (%) (%) n 11 11 11 11 1111 11 11 11 Mean 52.455 3.106 10.177 6.727 5.115 0.821 0.926 34.51838.991 Std Dev 9.1674 1.8231 2.3315 2.0089 1.8975 0.1387 0.1787 4.92506.6636 % CV 17.4768 58.6897 22.9088 29.8623 37.0944 16.8921 19.294014.2678 17.0900 Median 54.200 3.090 9.870 7.070 5.720 0.840 0.970 33.60040.200 Minimum 40.00 1.09 7.28 3.72 2.06 0.61 0.62 27.80 30.30 Maximum67.40 6.77 14.70 10.30 8.21 1.02 1.17 43.40 51.80 Geo.mean 51.715 2.6219.938 6.447 4.761 0.810 0.910 34.202 38.486 Footnotes: Note: PKparameter values were determined by 2-compartmental method. Geo. Mean =Geometric Mean [1] C168 = FIX activity above baseline at 168 hr afterdose. [2] TBLP1 = Estimated time after dose when FIX activity hasdeclined to 1 IU/dL above baseline. [3] TBLP3 = Estimated time afterdose when FIX activity has declined to 3 IU/dL above baseline. [4] TBLP5= Estimated time after dose when FIX activity has declined to 5 IU/dLabove baseline. [5] Incremental Recover was calculated using modelpredicted Cmax value generated from background subtracted resultsdivided by dose. [6] Incremental Recovery was calculated using theobserved maximum post-dose sample result; Incremental Recovery =(Baseline Subtracted Cmax observed)/Dose. [7] In-vivo Recovery = 100 ×(Model Predicted Cmax from baseline subtracted data/Dose) × PlasmaVolume (dL)/Dose in IU; where plasma volume in mL = (23.7 × Ht in cm) +(9.0 × Wt in kg) − 1709. [8] In-vivo Recovery = 100 × (BaselineSubtracted Observed Cmax) × Plasma Volume (dL)/Dose in IU; where plasmavolume in mL = (23.7 × Ht in cm) + (9.0 × Wt in kg) − 1709.

TABLE 14 Mean PK Values Based on Antigen Level Nominal Actual EquivalentAlpha Beta Dose Dose Dose C_(max) AUC_(INF) Cl V_(SS) MRT HL HL (IU/kg)(IU/kg) (mg/kg) Patient (ng/mL) (h*ng/mL) (mL/h/kg) (mL/kg) (h) (h) (h)12.5 13.714 0.228 1 1670 91300 2.5  245 98.2 21.2 107   25 27.25 0.453 22730 144000 3.14 273 87.1 11.3 71  50 54.5 0.905 3 5470 356000 2.54 366144   18.6 138   54.5 0.905 4 6910 389000 2.32 244 105   10.6  85.3 54.50.905 5 7520 416000 2.17 184 84.5 NC  94.3 54.513 0.906 6 11700 5310001.71 190 112   NC 140   55.878 0.928 7 5950 348000 2.67 310 116   10.1 93.9 100 109 1.81 10 12500 667000 2.72 263 96.8  9.79 78  109 1.81 821600 1200000 1.51 156 103   15.7  94.3 109 1.81 9 13400 998000 1.81 248137   11.5 107   109.176 1.81 11 17200 844000 2.15 226 105   13    97.1109.441 1.82 12 12500 778000 2.34 295 126   10.6 102   N 12 12 12    1212   12   12  Mean 9929.0 563525.0 2.3  250.0  109.6^(b )  13.2^(c) 100.7^(e) SD 5940.0 339925.0  0.5^(a ) 58.2 18.5   4.0^(d)  20.9 SE1715.0 98128.0 0.1  16.8  5.3  1.3  6.0 Geometric Mean 8614.0 452356.02.3  243.7 108.2  12.8  98.8 Due to correction of rounding or othererrors, ^(a)should be 0.46, ^(b)should be 110, ^(c)should be 12.0,^(d)should be 3.95, and ^(e)should be 101.

TABLE 15 Biochemical characterization of Factor IX Gamma-carboxylationFIXFc rFIX pdFIX aa 1-23 % 6 Gla 97.8 96.9 99.6 (K1K2 peptide) % 5 Gla2.2 3.1 0.4 % 4 Gla 0 0 0 aa 24-43 % 6 Gla 61.3 63.7 98.9 (K3 peptide) %5 Gla 26.3 30.9 1.1 % 4 Gla 12.5 5.4 0 Total Gla/mol, peptide map 11.511.6 12.0 Total Gla/mol, AAA 11.3 ± 0.3 11.5 ± 0.3 (12) Propeptidecontent none detected none detected none detected β-hydroxylation Asp 6470% 49%  37% Sulfation of Tyr 155  4%  5% (>90%) Phosphorylation of Ser158 <10%  <10%  (>90%) Ala 148/Thr 148 0/100% 100/0% 30/70% ActivatedFIX 0.0125%    0.109 +/− 0.00185% 0.21 +/− 0.010% FXIa Activation 94.8+/− 2.4%    96.6 +/− 1.8% Not done

TABLE 16 Summary of terminal half-lives of FIXFc and BENEFIX ™ after asingle intravenous dose. Species BENEFIX ™ FIXFc Normal mice 12.3 hr47.2 ± 4.8 hr FIX-deficient mice 13.2 hr 46.2 ± 10.1 hr FcRN KO mice16.5 ± 3.0 hr 16.9 ± 2.1 hr FcRN Tg32b mice 14.2 ± 2.9 hr 53.0 ± 6.6. hrRats 5.8 hr 34.8 ± 5.3 hr FIX-deficient dogs 14-18 hr * 47.5 hr Monkey12.7 hr ^(†) 47.3 ± 9.1 hr * Brinkhous et al, Blood, 1996; 88: 2603-2610^(†) McCarthy et al, 2002, Thromb Haemost, 2002; 87: 824-830

TABLE 17 Summary of in vitro ROTEM ® parameters for rFIXFc and BENEFIX ™spiked in pooled HemB mouse whole blood % of Alpha Angle Normal CT (sec)CFT (sec) (°) Activity (Mean ± SD) (Mean ± SD) (Mean ± SD) rFIXFc 0.074 2263 ± 209 1152 ± 170 24 ± 5 (n = 10 pools 0.74 1371 ± 82 459 ± 45 34 ±5 7.4 790.8 ± 30  226 ± 20 52 ± 2 BENEFIX ™ 0.1  2019 ± 178  732 ± 12330 ± 3 (n = 10 pools) 1 1090 ± 38 324 ± 33 43 ± 3 10 551.1 ± 38  127 ±10 67 ± 2

TABLE 18 Median blood loss following tail clip in HemB mice treated withrFIXFc or BENEFIX ™ Median Blood Loss (mL) rFIXFc BENEFIX ™ Vehicle Dose(IU/kg) (n = 15/dose) (n = 15/dose) (n = 18) 720 0.101 360 0.651 0.218240 0.298 120 0.4567 0.564 80 0.8474 40 1.0097 0.918 0 1.1586

TABLE 19 Ex vivo ROTEM ® parameter in HemB mice treated with rFIXFc andBENEFIX ™ Alpha Angle Time CT (sec) CFT (sec) (degree) (hour) (Mean ±SD) (Mean ± SD) (Mean ± SD) 100 IU/kg 0.083 599 ± 23 174 ± 16 58 ± 2BENEFIX ™ 24 682 ± 49 184 ± 34 57 ± 5 (n = 4 mice/ 48  897 ± 114 310 ±89 45 ± 7 time point) 72 1141 ± 155  508 ± 123 32 ± 7 96 1613 ± 181 605± 92 27 ± 3 50 IU/kg 0.083 700 ± 18 213 ± 9  53 ± 1 rFIXFc 24 836 ± 31261 ± 15 47 ± 2 (n = 8 mice/ 72 845 ± 38 285 ± 17 45 ± 2 time point) 96957 ± 30 296 ± 26 43 ± 2 120 1014 ± 83  342 ± 50 42 ± 4 168 1139 ± 65 408 ± 41 36 ± 3 216 1366 ± 96  453 ± 48 34 ± 3

TABLE 20A PK parameters of rFIXFc and BENEFIX ™ (200 IU/kg) followingsubcutaneous injection of a single dose in FIX-deficient mice (AntigenELISA) Absorp- Elimina- tion tion AUC_(INF)/ Dose V/F Tlag AUC_(INF) HLHL CL/F Tmax Cmax Dose Cmax/Dose F Compound ng/kg mL/kg Hr Hr*ng/mL HrHr mL/Hr/kg Hr ng/mL Hr.kg/mL g/mL % BeneFIX 727273 3920 2.86 6397 1.9623.9 114 10.6 148 0.00880 0.204 23.3 rFIXFc 3278689 2071 0.896 1413707.67 61.9 23.2 27.3 1178 0.0431 0.359 381

TABLE 20B PK parameters of rFIXFc and BENEFIX ™ (200 IU/kg) followingsubcutaneous injection of a single dose in FIX-deficient mice (aPTTactivity assay) Absorp- Elimina- tion tion AUC_(INF)/ Dose V/F TlagAUC_(INF) HL HL CL/F Tmax Cmax Dose Cmax/Dose F Compound IU/kg dL/kg HrHr*IU/dL Hr Hr dL/Hr/kg Hr IU/dL Hr*kg/dL g/dL % BeneFIX 207 54.8 0.63193.9 7.01 17.2 2.20 16.0 2.04 0.454 9.86 18.9 rFIXFc 172 25.1 2.32 4186.84 42.4 0.411 23.8 4.82 2.43 28.0 29.1

TABLE 21 PK and PD Analysis of rFIXFc and BENEFIX ™ After a SingleSubcutaneous Dose in FIX-Deficient Mice. AUC/Dose Elim. Half- CL/F TmaxCmax/Dose Assay (Hr*kg/mL) Life (Hr) (mL/Hr/kg)/% (Hr) (kg/mL) F (%)rFIXFc 200 IU/kg Antigen 0.041 61.9 23.2 27.3 0.00035 38.1 BENEFIX ™ 200Antigen 0.0073 23.9 114 10.6 0.00017 23.3 IU/kg Ratio Antigen 5.62 2.590.20 2.58 2.05 1.63 (rFIXFc/BENEFIX ™) rFIXFc 400 IU/kg Antigen 0.04250.9 23.7 18.3 0.00045 45.6 BENEFIX ™ 400 Antigen 0.0089 20.2 113 8.130.00024 20.2 IU/kg Ratio Antigen 4.72 2.52 0.21 2.25 1.91 2.26(rFlXFc/ENEFIX ™) AUC/Dose Elim. Half- CL/F Tmax Cmax/Dose (Hr*kg/mL)Life (Hr) (mL/Hr/kg)/% (Hr) (kg/mL) F (%) rFIXFc 200 IU/kg Activity0.021 42.4 41.1 23.8 0.00024 29.1 BENEFIX ™ 200 Activity 0.0047 17.2 22016.0 0.00010 18.9 IU/kg Ratio Activity 4.47 2.46 0.19 1.49 2.40 1.54(rFIXFc/BENEFIX ™) rFIXFc 400 IU/kg Activity 0.028 40.3 35.6 15.90.00037 39.2 BENEFIX ™ 400 Activity 0.0052 15.6 193 18.1 0.00010 15.5IU/kg Ratio Activity 5.38 2.58 0.18 0.88 3.70 2.53 (rFIXFc/BENEFIX ™)

TABLE 22 PK parameters of rFIXFc (50 IU/kg) following subcutaneousinjection of a single dose in cynomolgus monkeys. V/F AUC AbsorptionTerminal CL/F Tmax Cmax AUC/D Group Animal_ID (mL/kg) (Hr*ng/mL) HL (Hr)HL (Hr) (mL/Hr/kg) (Hr) (ng/mL) (Hr*kg/mL) F(%) 50 5C4 545 109000 8.4250.2 7.53 26.1 1050 0.133 43.7 IU/kg C37716 975 108000 6.4 89 7.6 26.2685 0.132 43.3 rFIXFc C41440 622 82500 8.54 43.4 9.93 24.9 885 0.10133.1 N 3 3 3 3 3 3 3 3 3 Mean 714 99800 7.79 60.9 8.35 25.7 873 0.12240.1 SD 229 14900 1.2 24.6 1.37 0.685 182 0.0183 6.03 SE 132 8630 0.69514.2 0.79 0.396 105 0.0106 3.48 Geometric 691 99000 7.72 57.9 8.28 25.7860 0.121 39.7 Mean CV % 31.2 15.8 16.4 39.4 15.8 2.68 21.7 15.9 15.9Geometric Mean

TABLE 23 PK parameters of rFIXFc (100 IU/kg) following subcutaneousinjection of a single dose in cynomolgus monkeys. V/F AUC AbsorptionTerminal CL/F Tmax Cmax AUC/D Group Animal_ID (mL/kg (Hr*ng/mL) HL (Hr)HL (Hr) (mL/Hr/kg) (Hr) (ng/mL) (Hr*kg/mL) F(%) 100 29109 1630 6980011.4 48.1 23.5 31 644 0.0426 14.0 IU/kg 605097 561 207000 5.12 49 2 7.918:6 2250 0.126 41.5 rFIXFc C35785 387 238000 6.37 39 6.89 19.9 29700.145 47.8 N 3 3 3 3 3 3 3 3 3 Mean 858 172000 7.62 45.4 12.8 23.2 19500.105 34.4 SD 671 89600 3.31 5.58 9.3 6.79 1190 0.0546 18.0 SE 388 517001.91 3.22 5.37 3.92 687 0.0315 10.4 Geometric 707 151000 7.18 45.2 10.922.6 1630 0.0921 30.3 Mean CV % 86.2 75.5 43.1 12.8 75.5 28.2 96.9 75.575.5 Geometric Mean

TABLE 24 PK parameters of rFIXFc (200 IU/kg) following subcutaneousinjection of a single dose in cynomolgus monkeys. V/F AUC AbsorptionTerminal CL/F Tmax Cmax AUC/D Group Animal_ID (mL/kg) (Hr*ng/mL) HL (Hr)HL (Hr) (mL/Hr/kg) (Hr) (ng/mL) (Hr*kg/mL) F(%) 200 50883 855 4080003.36 73.7 8.03 15.7 3310 0.124 40.9 IU/kg C31129 461 415000 6.42 40.47.91 20.2 5030 0.127 41.6 rFIXFc C41410 147 262000 11.5 32.6 3.12 26.73160 0.0799 26.3 N 3 3 3 3 3 3 3 3 3 Mean 487 362000 7.08 48.9 6.36 20.93830 0.110 36.3 SD 354 86100 4.06 21.8 2.8 5.51 1040 0.0263 8.67 SE 20549700 2.36 12.6 1.62 3.16 598 0.0152 5.00 Geometric 387 354000 6.27 465.83 20.4 3750 0.108 35.5 Mean CV % 110 26.4 67.6 44.2 58.3 27 25.9 26.526.5 Geometric Mean

TABLE 25 PK Analysis of rFIXFc Following a Single Subcutaneous Dose inCynomolgus Monkeys AUC Abs Elim. CL/F rFIXFc (Hr*ng/ Half- Half- (mL/Hr/Tmax Cmax (IU/kg) mL) Life (Hr) Life (Hr) kg)/% (Hr) (ng/mL) F(%) 50Geo. 99,000 7.72 57.9 8.28 25.7 860 39.7 Mean CV % 15.8 16.4 39.4 15.82.68 21.7 15.9 Geo. Mn 100 Geo. 221,959 5.71 43.8 7.38 19.2 2,585 44.5Mean CV % 9.89 15.5 16.5 9.70 4.78 19.8 10.0 Geo. Mn 200 Geo. 354,0006.27 46 5.83 20.4 3,750 35.5 Mean CV % 26.4 67.6 44.2 58.3 27 25.9 26.5Geo. Mn Bioavailability ranged from 35.5 to 44.5% for rFIXFc Eliminationhalf-life ranged 43.8 to 57.9 hrs for rFIXFc

TABLE 26 Dosing Guidelines For rFIXFc Therapy In Hemophilia B Factor IXFrequency Level Required of Doses Type of Hemorrhage (%) (hrs) MinorEpistaxis Hemarthroses, uncomplicated 20-30 48 Superficial muscular20-30 48 Superficial soft tissue 20-30 48 Moderate EpistaxisIntramuscular with dissection 25-50 48 Soft tissue with dissection 25-5048 Mucous membranes 25-50 48 Dental extractions 25-50 48 Hematuria 25-5048 Hemarthroses, with limited motion 40-80 48 Major Epistaxis  50-10024-48 Pharynx  50-100 24-48 Retropharynx  50-100 24-48 Retroperitoneum 50-100 24-48 Surgery  50-100 24-48 CNS  50-100 24-48 Patient shouldconsult with the their physicians, but should take only 1 follow-up dosenot less than 24-48 hours after the initial dose.Tables 27A and 27B. Comparison of data using calculations in (A) Example1 and (B) Example 11.A

Parameter (mean ± SD) Incremental Time to 1% Recovery above Dose C_(max)AUC_(INF) CL Vss (IU/dL per C_(168h) baseline (IU/kg) n (IU/dL) (h ·IU/dL) (mL/h/kg) (mL/kg) MRT (h) T1/2α (h) T1/2β (h) IU/kg)* (IU/kg)^(†)(Day)^(‡) 25 1 19.9 753 3.62 275 76.0 1.20 54.0 0.77 1.09 7.28 50 5 41.6± 8.97/ 1630 ± 750 3.77 ± 1.12  264 ± 77.6 71.7 ± 13.0 1.79 ± 1.19 52.1± 10.4 0.86 ± 0.22 2.23 ± 1.40 9.29 ± 1.98 8.98 100 5 98.2 ± 8.21/ 3930± 893 2.89 ± 0.615 179 ± 31.1 62.8 ± 8.82 9.99 ± 4.99 52.5 ± 10.1 1.02 ±0.11 4.38 ± 1.53 11.6 ± 1.97 8.23 25-100 11 NA^(§) NA^(§) ND ND ND ND NDND NA^(§) NA^(§)B

Parameter (mean ± SD) (Range) Dose C_(max) AUC_(INF) CL Vss (IU/kg) n(IU/dL) (h · IU/dL) (mL/h/kg) (mL/kg) MRT (h) 25 1 20.4 766 3.56 27176.2 50 5 47.5 ± 12.8 1700 ± 550 3.44 ± 0.84 262 ± 55.4 76.8 ± 6.7 (33.0-61.1)  (1300-2660) (2.05-4.18) (163-296)  (67.9-85.9) 100 5 98.5 ±7.9  4020 ± 986 2.84 ± 0.66 183 ± 27.9 65.9 ± 10.3 (90.8-110)  (3090-5130) (2.13-3.55) (162-221)  (53.2-76.5) 25-100 11 NA^(§) NA^(§)3.18 ± 0.78 227 ± 58.6 71.8 ± 10.0 (2.05-4.18) (162-296)  (53.2-85.9)Parameter (mean ± SD) (Range) Incremental Time to 1% Recovery above Dose(IU/dL per C_(168h) baseline (IU/kg) T1/2α_(□)(h) T1/2β (h) IU/kg)*(IU/dL)^(†) (Day)^(‡) 25 0.61 53.5 0.77 1.11 7.3 50 3.4 ± 3.4 57.5 ±8.2  0.87 ± 0.21 2.46 ± 0.89 10.1 ± 1.5 (0.13-8.72) (47.9-67.2)(0.63-1.12) (1.63-3.92)  (8.4-12.3) 100 10.3 ± 5.6  56.5 ± 14.1 1.02 ±0.11 4.65 ± 1.73 12.3 ± 2.5 (3.97-16.6) (42.4-74.5) (0.89-1.18)(3.08-6.85)  (9.9-15.0) 25-100 NA^(§) 56.7 ± 10.9 0.93 ± 0.18 NA^(§)NA^(§) (42.4-74.5) (0.63-1.18) Results presented are mean ± SD with,range listed in the parentheses C_(max) indicates maximum concentration;AUC_(INF), area under the curve (time zero extrapolated to infinitetime); CL, clearance; Vss, volume of distribution at steady state; MRT,mean residence time; T1/2□, distribution half-life; T1/2□, eliminationhalf-life; NA, not applicable *Incremental recovery was calculated usingobserved C_(max) subtracted with pretreatment baseline value and dividedby dose ^(†)Plasma FIX activity above baseline at 168 hours (7 days)post dose ^(‡)Model-predicted time after dose when FIX activity declinedto 1 IU/dL above subject's baseline ^(§)Data are not applicable becauseparameters are not dose-independent, thus the mean and SD values werenot calculated across the different dose groups

What is claimed is:
 1. A method of controlling the incidence of a bleeding episode in a subject having hemophilia comprising administering to the subject multiple doses of at least about 50 IU/kg of a long-acting Factor IX (“FIX”) polypeptide at a dosing interval of about a week or longer between two doses.
 2. The method of claim 1, wherein the long-acting FIX polypeptide comprises an FIX portion and an Fc portion and exhibits one or more characteristics selected from the group consisting of: a. an incremental recovery (K-Value) (activity; observed) greater than 0.75 IU/dL per IU/kg; b. an incremental recovery (K value) (activity; observed) of about 0.62-1.17 IU/dL per IU/kg; c. a clearance (CL) (activity) of about 1.84-4.58 mL/hour/kg; d. a clearance (CL) (activity) of about 3.36±0.93 mL/hour/kg; e. a mean residence time (MRT) (activity) of about 53.1-85.8 hours; f. a mean residence time (MRT) (activity) of about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, or about 90 hours; g. a MRT (activity) of at least about 68.05±11.16 hours; h. a t_(1/2beta) (activity) of about 40-67.4 hours; i. a t_(1/2beta) (activity) of at least about 40, about 45, about 50, about 55, about 60, about 65, about 70, or about 75 hours; j. a t_(1/2beta) (activity) of about 3 fold longer than the t_(1/2beta) (activity) of a polypeptide consisting of FIX; k. a Vss (activity) of about 145-365 mL/kg; l. a Vss (activity) of about 226±6736 mL/kg; m. an AUC/dose of about 21.80-54.30 IU*h/dL per IU/kg; and n. an AUC/dose of about 32.44±10.75 IU*h/dL per IU/kg.
 3. The method of claim 1, wherein each of said doses is selected from about 50 IU/kg to about 60 IU/kg, about 60 IU/kg to about 70 IU/kg, about 70 IU/kg to about 80 IU/kg, about 80 IU/kg to about 90 IU/kg, or about 90 IU/kg, to about 100 IU/kg.
 4. The method of claim 1, wherein each of said doses is selected from about 50 nag, about 55 IU/kg, about 60 IU/kg, about 65 IU/kg, about 70 IU/kg, about 75 IU/kg, about 80 IU/kg, about 85 IU/kg, about 90 III/kg, about 95 IU/kg, or about 100 IU/kg.
 5. The method of claim 1, wherein each of the multiple doses is about 50 IU/kg and the dosing interval is about a week.
 6. The method of claim 1, wherein each of the multiple doses is about 100 and the dosing interval is 10 days.
 7. The method of claim 2, wherein the FIX portion is at least 90% identical to amino acids 1 to 415 of SEQ ID NO:2.
 8. The method of claim 2, wherein the Fe portion is at least 90% identical to amino acids 1 to 227 of SEQ ID NO:4.
 9. The method of claim 2, wherein the long-acting FIX polypeptide is in the form of a hybrid comprising a second polypeptide, which comprises an additional Fe portion associated with the first Fe portion.
 10. The method of claim 1, wherein less than 25% of FIX in the long-acting FIX polypeptide in said doses are fully phosphorylated and less than 25% of FIX in the long-acting FIX polypeptide in said doses are fully sulfated.
 11. The method of claim 1, wherein said long-acting FIX polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient.
 12. The method of claim 1, wherein said doses are administered intravenously or subcutaneously.
 13. The method of claim 1, wherein the dosing interval is about 10 days or longer.
 14. The method of claim 1, which is for prophylactic treatment of hemophilia B.
 15. The method of claim 1, wherein the dosing interval is about a week to about 10 days.
 16. The method of claim 1, which is for on-demand treatment.
 17. The method of claim 16, wherein each of the multiple doses is about 50 IU/kg to about 100 IU/kg.
 18. The method of claim 1, wherein the plasma level of the long-acting FIX polypeptide reaches an average trough of at least about 1 IU/dl.
 19. A method of controlling a bleeding episode in a hemophilia patient comprising administering to the patient multiple doses of at least about 50 IU/kg of a long-acting FIX polypeptide at a dosing interval of about a week between two doses, wherein the long-acting FIX polypeptide comprises a FIX portion and an FcRn Binding Partner portion.
 20. The method of claim 19, wherein the FcRn Binding Partner portion comprises an Fc portion.
 21. A method of controlling a bleeding episode in a hemophilia patient comprising administering to the patient multiple doses of at least about 100 IU/kg of a long-acting FIX polypeptide at a dosing interval of about 10 days between two doses, wherein the long-acting FIX polypeptide comprises a FIX portion and an FcRn Binding Partner portion.
 22. The method of claim 21, wherein the FcRn Binding Partner portion comprises an Fc portion. 